Systems and methods for multiple analyte detection

ABSTRACT

Systems and methods for multiple analyte detection include a system for distribution of a biological sample that includes a substrate, wherein the substrate includes a plurality of sample chambers, a sample introduction channel for each sample chamber, and a venting channel for each sample chamber. The system may further include a preloaded reagent contained in each sample chamber and configured for nucleic acid analysis of a biological sample that enters the substrate and a sealing instrument configured to be placed in contact with the substrate to seal each sample chamber so as to substantially prevent sample contained in each sample chamber from flowing out of each sample chamber. The substrate can be constructed of detection-compatible and assay-compatible materials.

CROSS-REFERENCE TO COPENDING APPLICATIONS

This application is a continuation of patent application Ser. No.11/380,327 filed Apr. 26, 2006, which claims the benefit of priority toU.S. Provisional Application No. 60/674,750 filed on Apr. 26, 2005, bothof which are incorporated by reference in their entirety herein.

This application makes cross-reference to U.S. Provisional ApplicationNo. 60/674,876, entitled “System for Population Security andEpidemiological Analysis,” concurrently filed with U.S. ProvisionalApplication No. 60/674,750, and later filed 60/696,157 of the sametitle, all of which are also incorporated by reference herein in theirentirety.

FIELD

The present teachings relate to systems and methods for multiple analytedetection.

BACKGROUND

Biochemical testing for research and diagnostic applications can requiresimultaneous assays including a large number of analytes in conjunctionwith one or a few samples and can include extended sample manipulation,multiple test substrates, multiple analytical instruments, and othersteps. It is desirable to provide a method for analyzing one or a fewbiological samples using a single test device with a large number ofanalytes while requiring a small amount of sample. It is desirable toprovide a device that is small in size while providing high-sensitivitydetection for the analytes of interest with minimal sample manipulation.It is desirable to provide a method of loading the sample(s) intochambers on the substrate and individually sealing each chamber. It isfurther desirable to provide a mechanism for venting of the substrateduring filling, while also avoiding and/or minimizing leakage of fluid(e.g., biological sample and/or reagents) from the test device.

SUMMARY

In various embodiments, the present teachings can provide a system fordistribution of a biological sample including a substrate with aplurality of sample chambers, a sample introduction channel for eachsample chamber, and a venting channel for each sample chamber, whereinthe substrate is constructed of detection-compatible andassay-compatible materials, and a sealing plate with sealing protrusionsfor sealing the sample introduction channels and the venting channelsfor each sample chamber.

In various embodiments, the present teachings can provide a method fordistribution of a biological sample including providing an injectionmolded base, wherein the base includes a plurality of sample cavities, asample introduction trench for each sample cavity, and a venting trenchfor each sample cavity, and wherein the base is constructed ofdetection-compatible and assay-compatible materials, providing a film toadhere to the base forming a substrate, wherein film includes aplurality of vents, and wherein the film forms a plurality of samplechambers from each sample cavity, a sample introduction channel for eachsample introduction trench, and a venting channel for sample ventingtrench, providing the biological sample to the substrate, forcing thebiological sample to the sample chambers through the sample introductionchannels, providing a sealing plate comprising sealing protrusions foreach sample chamber, heating the sealing plate, sealing the samplechambers by contacting the sealing protrusions with the sampleintroductory channels and the venting channels of each sample chamber.

In various embodiments, the present teachings may provide a system fordistribution of a biological sample that includes a substrate, whereinthe substrate includes a plurality of sample chambers, a sampleintroduction channel for each sample chamber, and a venting channel foreach sample chamber. The system may further include a preloaded reagentcontained in each sample chamber and configured for nucleic acidanalysis of a biological sample that enters the substrate, and a sealinginstrument configured to be placed in contact with the substrate to sealeach sample chamber so as to substantially prevent sample contained ineach sample chamber from flowing out of each sample chamber. Thesubstrate can be constructed of detection-compatible andassay-compatible materials.

In still further various embodiments, a method for distribution of abiological sample may include providing a base, wherein the baseincludes a plurality of sample cavities containing a preloaded reagenttherein, a sample introduction trench for each sample cavity, and aventing trench for each sample cavity, and wherein the base isconstructed of detection-compatible and assay-compatible materials. Themethod also may include providing a film to adhere to the base to form asubstrate, wherein the film and base form a plurality of sample chambersfrom each sample cavity, a sample introduction channel from each sampleintroduction trench, and a venting channel from each sample ventingtrench. The method may further include supplying the biological sampleto the substrate, filling the sample chambers with the biological samplevia the sample introduction channels, passing gas out of the substratevia at least one venting mechanism in the substrate, and sealing thesample chambers to substantially prevent sample in the sample chambersfrom flowing out of the sample chambers.

According to yet other embodiments, the present teachings may provide asystem for distribution of a biological sample that includes means fordistributing the sample to a plurality of sample chambers containing apreloaded reagent, means for venting each of the sample chambers, andmeans for sealing each of the sample chambers.

In various embodiments, the present teachings also can provide a devicefor testing a biological sample that includes a substrate defining aplurality of distribution portions configured to distribute biologicalsample throughout the substrate, the plurality of distribution portionscomprising a plurality of sample chambers, a sample introduction channelfor each sample chamber, and a venting channel for each sample chamber.The device also may include a substance disposed in at least one of thedistribution portions, the substance being configured to seal eachsample chamber so as to substantially prevent sample disposed in eachsample chamber from flowing out of each sample chamber during testing ofthe biological sample.

Additional embodiments are set forth in part in the description thatfollows, and in part will be apparent from the description, or may belearned by practice of the various embodiments described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the present teachings are exemplified in theaccompanying drawings. The teachings are not limited to the embodimentsdepicted, and include equivalent structures and methods as set forth inthe following description and known to those of ordinary skill in theart. In the drawings:

FIG. 1 illustrates a perspective view of a substrate base for biologicalanalysis according to various embodiments of the present teachings;

FIG. 2A illustrates a top view of a substrate for biological analysisaccording to various embodiments of the present teachings

FIG. 2B illustrates an exploded perspective view of a substrate forbiological analysis according to various embodiments of the presentteachings;

FIGS. 3A-3B illustrate a perspective view with magnified section of asealing plate according to various embodiments of the present teachings;

FIG. 3C is a partial perspective view of another sealing plate andsubstrate according to various embodiments of the present teachings;

FIG. 3D is a partial view of the sealing plate of FIG. 3C from theprotrusion side of the plate;

FIG. 4 illustrates an exploded perspective view of a substrate forbiological analysis with a sealing plate according to variousembodiments of the present teachings;

FIG. 5 illustrates top view of a substrate for biological analysisaccording to various embodiments of the present teachings;

FIG. 6 illustrates a perspective view of an instrument for biologicalsample preparation including six fluidic cartridges according to variousembodiments of the present teachings;

FIG. 7 illustrates a cut-away perspective view of a cartridge forbiological sample preparation according to various embodiments of thepresent teachings;

FIG. 8A illustrates a perspective view of a substrate for biologicalanalysis according to various embodiments of the present teachings;

FIGS. 8B and 8C illustrate cross-sectional views of FIG. 8A takenthrough line 8C-8C according to various embodiments of the presentteachings;

FIGS. 9A-9B illustrate perspective views of exemplary embodiments forsealing the substrate of FIGS. 8A-8C and 10A-10C;

FIG. 10A illustrates a perspective view of a substrate for biologicalanalysis according to various embodiments of the present teachings;

FIGS. 10B and 10C illustrate cross-sectional views of FIG. 10A takenthrough line 10C-10C according to various embodiments of the presentteachings;

FIG. 11 illustrates a cross-sectional view of a substrate for biologicalanalysis according to various embodiments of the present teachings;

FIGS. 12A-12B are cross-sectional views of filling and sealing thesubstrate of FIG. 11 according to various embodiments of the presentteachings;

FIG. 13 is a perspective view of a substrate for biological analysisaccording to various embodiments of the present teachings;

FIG. 14 is a perspective, isometric view of another substrate forbiological analysis according to various embodiments of the presentteachings;

FIG. 15 is a perspective, isometric view of yet another substrate forbiological analysis according to various embodiments of the presentteachings;

FIG. 16 is a perspective, isometric view of a substrate for biologicalanalysis according to various embodiments of the present teachings;

FIG. 17 is a perspective, isometric view of yet another substrate forbiological analysis according to various embodiments of the presentteachings;

FIGS. 18A-18C illustrate perspective views of vent holes formed invarious materials via Oxford Laser, Inc. instruments;

FIG. 19 is a perspective view of yet another substrate for biologicalanalysis according to various embodiments of the present teachings;

FIGS. 20A and 20B illustrate perspective views of sample chambers,venting channels, and venting chambers according to various embodimentsof the present teachings;

FIGS. 21A-21C schematically illustrate cross-sectional views ofexemplary steps of filling a feature of a substrate for biologicalanalysis;

FIGS. 22A-22C schematically illustrate cross-sectional views ofexemplary steps of filling a feature of a substrate for biologicalanalysis according to various embodiments of the present teachings;

FIGS. 23A and 23B schematically illustrate cross-sectional views ofexemplary steps of filling a feature of a substrate for biologicalanalysis;

FIG. 24 illustrates a top view of yet another substrate for biologicalanalysis according to various embodiments of the present teachings;

FIG. 25 illustrates a perspective view of yet another substrate forbiological analysis according to various embodiments of the presentteachings;

FIG. 26 illustrates a top view of yet another substrate for biologicalanalysis according to various embodiments of the present teachings;

FIG. 27 illustrates a top view of yet another substrate for biologicalanalysis according to various embodiments of the present teachings;

FIG. 28 is a partial cross-sectional view of a sample chamber andventing chamber provided with a vent through hole according to variousembodiments of the present teachings;

FIGS. 29 and 30 are top and perspective views of a sealing roller forsealing a substrate according to various embodiments of the presentteachings;

FIG. 31 is a perspective view of another sealing roller according tovarious embodiments of the present teachings;

FIGS. 32 and 33 are partial, cross-sectional views of a substrate and athermal block for sealing a substrate according to various embodimentsof the present teachings;

FIGS. 34, 35, and 35A are partial, cross-sectional views of thermalblocks for sealing a substrate according to various embodiments of thepresent teachings;

FIG. 36 is a top view of a sample chamber with an escape channelaccording to various embodiments of the present teachings;

FIG. 37 is a partial cross-sectional view of a substrate that usescapacitance overfill detection in accordance with various embodiments ofthe present teachings;

FIG. 38 is a perspective view of a substrate for biological analysisaccording to various embodiments of the present teachings;

FIGS. 39A-39C are perspective views of steps of filling and sealing thesubstrate of FIG. 38 according to various embodiments of the presentteachings;

FIG. 40A is a partial, perspective, isometric view of a substrate forbiological analysis according to various embodiments of the presentteachings;

FIGS. 40B and 40C are partial cross-sectional views of the substrate ofFIG. 40A showing the substrate before and after being filled withsample, respectively;

FIGS. 41A and 41B are partial perspective views of a substrate forbiological analysis according to various embodiments of the presentteachings;

FIGS. 42A-42C are partial perspective views of a substrate forbiological analysis according to various embodiments of the presentteachings;

FIGS. 43A-43C are partial perspective views of a substrate forbiological analysis according to various embodiments of the presentteachings;

FIG. 44 is a perspective view of a substrate for biological analysisaccording to various embodiments of the present teachings;

FIG. 44A is a close-up of section 44A of the substrate of FIG. 44;

FIG. 45 is a perspective view of a device for inserting porous fibers insubstrates for biological analysis according to various embodiments ofthe present teachings;

FIGS. 46A-46D show exemplary steps for making the substrate of FIG. 44;

FIGS. 47A and 47B are a partial perspective and cross-sectional view ofa substrate for biological analysis according to various embodiments ofthe present teachings;

FIGS. 48A-48C are perspective views of yet another substrate forbiological analysis according to various embodiments of the presentteachings;

FIG. 49 is a partial cross-sectional view of the substrate of FIGS.48A-48C;

FIG. 50 is a partial perspective view of the substrate of FIGS. 48A-48C;

FIGS. 51A and 51B are perspective views of yet another substrate forbiological analysis according to various embodiments of the presentteachings;

FIG. 52 is a perspective view of a system for packaging a plurality ofsubstrates for biological analysis according to various embodiments ofthe present teachings;

FIGS. 53A and 53B are a perspective and cross-sectional view of athermocycler in accordance with various embodiments of the presentteachings;

FIG. 54 is a perspective view of a holding fixture and subcards inaccordance with various embodiments of the present teachings;

FIG. 55 is a perspective view of a holding fixture and subcards inaccordance with various embodiments of the present teachings;

FIG. 56 is a perspective view of another holding fixture and subcards inaccordance with various embodiments of the present teachings; and

FIGS. 57-61 are perspective views of substrate carriers in accordancewith various embodiments of the present teachings.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide a further explanation of the variousembodiments of the present teachings.

DESCRIPTION OF VARIOUS EMBODIMENTS

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise. Wherever possible, the same reference numbers will beused throughout the drawings to refer to the same or like parts.

The section headings used herein are for organizational purposes only,and are not to be construed as limiting the subject matter described.All documents cited in this application, including, but not limited topatents, patent applications, articles, books, and treatises, areexpressly incorporated by reference in their entirety for any purpose.

The term “sample chamber” as used herein refers to any structure thatprovides containment to a sample. The chamber can have any shapeincluding circular, rectangular, cylindrical, etc. Multi-chamber arrayscan include 12, 16, 24, 36, 48, 96, 192, 384, 3072, 6144, or more samplechambers. The term “channel” as used herein refers to any structure thatis smaller than a chamber. A channel can have any shape. It can bestraight or curved, as necessary, with cross-sections that are shallow,deep, square, rectangular, concave, or V-shaped, or any otherappropriate configuration. A “distribution portion” of a substrate mayrefer to any portion of the substrate configured to contain, flow,receive, or otherwise hold sample and/or gas in the substrate. Examplesof distribution portions include main fluid supply channels, ventingchannels, venting chambers, sample chambers, sample introductionchannels, overfill chambers, and virtually any other channels and/orchambers of the substrate.

The term “biological sample” as used herein refers to any biological orchemical substance, typically in an aqueous solution with luminescentdye that can produce emission light in relation to nucleic acid presentin the solution. The biological sample can include one or more nucleicacid sequence(s) to be incorporated as a reactant in a polymerase chainreaction (PCR) and other reactions such as ligase chain reactions,antibody binding reactions, oligonucleotide ligations assay,hybridization assays, and invader assays (e.g., for an isothermreaction). The biological sample can include one or more nucleic acidsequences to be identified for DNA sequencing.

The term “luminescent dye” as used herein may refer to fluorescent orphosphorescent dyes that can be excited by excitation light orchemiluminscent dyes that can be excited chemically. As used herein,“luminescent dye” may also include the use of energy transfer pairs(intramolecular or intermolecular). For example, excitation of oneenergy transfer pair member and emission of the other member may expandthe range of emission wavelengths for multiplexing. Quenchers selectedshould be suitable for both members of the energy transfer pair.Luminescent dyes can be used to provide different colors depending onthe dyes used. Several dyes will be apparent to one skilled in the artof dye chemistry, including, for example, intercalating dyes. One ormore colors can be collected for each dye to provide identification ofthe dye or dyes detected. The dye can be a dye-labeled fragment ofnucleotides. The dye can be a marker triggered by a fragment ofnucleotides. The dye can provide identification of a nucleic acidsequence in the biological sample by association, for example, bondingto or reacting with a detectable marker, for example, a respective dyeand quencher pair. The respective identifiable component can bepositively identified by the luminescence of the dye. The dye can benormally quenched, and then can become unquenched in the presence of aparticular nucleic acid sequence in the biological sample. Thefluorescent dyes can be selected to exhibit respective and, for example,different, excitation and emission wavelength ranges. The luminescentdye can be measured to quantitate the amount of nucleic acid sequencesin the biological sample. The luminescent dye can be detected inreal-time to provide information about the identifiable nucleic acidsequences throughout the reaction. Examples of fluorescent dyes withdesirable excitation and emission wavelengths can include 5-FAM™, TET™,and VIC™. The term “luminescence” as used herein refers tolow-temperature emission of light including fluorescence,phosphorescence, electroluminescence, and chemiluminescence.

In various embodiments, sample chambers can be dimensioned to hold from0.0001 μL to 10 μL of sample per chamber, or between 0.001 μL and 2 μL.Conveniently, the volume of each detection chamber is between 0.001 μLand 1 μL. For example, a chamber having a volume of 0.2 μL may havedimensions of 1 mm×1 mm×0.2 mm, where the last dimension is the chambersdepth. As a further example, a chamber may have a substantiallycylindrical shape having a diameter of about 1.96 mm and a depth ofabout 0.5 mm and a volume of about 1.35 μL.

In various embodiments, the sample introduction channels can bedimensioned to facilitate rapid delivery of sample to the samplechambers, while occupying as little volume as possible. For example,cross-sectional dimensions for the channels can range from 0.5 μm to 250μm for both the width and depth. In some embodiments, the channel pathlengths to the sample chambers can be minimized to reduce the totalchannel volume. For example, the network can be substantially planar,i.e., the sample introduction channels and sample chambers in thesubstrate intersect a common plane.

“Venting mechanisms” as used herein may refer to any mechanismconfigured and arranged to permit gas to escape therethrough and leavethe substrate. Venting mechanisms may be the last structure the gaspasses through prior to leaving the substrate or be a structure thatpasses the escaping gas therethrough to another structure that is thelast structure before the gas escapes the substrate. According tovarious embodiments, venting mechanisms may permit the passage of gaswhile substantially preventing the passage of liquid. Also, ventingmechanisms may be used in combination with one another to permit gas toescape the substrate. Examples of venting mechanisms include, but arenot limited to, gas-permeable or porous membranes, vent through holeseither in a film layer, a base or both, porous fibers, and hydrophobicliquid stops.

In various embodiments, there are a variety of means for distributingthe biological sample to the plurality of sample chambers. All of theseinclude applying a force. The force can be a pulling force or a pushingforce, depending on whether it provides a negative (pulling) or positive(pushing) force relative to the direction of fluid flow. Examples offorces and how the force is enacted upon the biological sample includespinning the substrate to provide centrifugal force to push the liquid,sizing the sample introductory channels to provide capillary force topull the liquid, aspirating the sample through the vents to pull theliquid, evacuating the sample chamber to pull the liquid, and/orproviding pressure, such as by pumping, compressing, plunging, etc. topush the liquid. In each of these configurations, the venting channelsand vents can be used to accommodate the displaced venting gas, whetherair or other gas such as nitrogen, that is pushed out by the sample orthe venting channels and vents can be used to evacuate the gas in thesample chambers to create a vacuum for the sample or aspirate sampleitself.

In various embodiments, the substrate that defines thesample-distribution network can be constructed from any solid materialthat is suitable for conducting analyte detection. Materials that can beused will include various plastic polymers and copolymers, such aspolypropylenes, polystyrenes, polyimides, COP, COC, and polycarbonates.Inorganic materials such as glass and silicon are also useful. Siliconis especially advantageous in view of its high thermal conductivity,which facilitates rapid heating and cooling of the substrate ifnecessary. The substrate can be formed from a single material or from aplurality of materials. Examples of this are described at U.S. Pat. No.6,126,899.

In various embodiments, the sample-distribution network includingcavities and trenches, for example, formed in a substrate base portion,can be formed by any suitable method known in the art. For plasticmaterials, injection molding can be suitable to form sample cavities andconnecting channels having a desired pattern. For silicon, standardetching, RIE, DRIE, and wet-etching techniques from the semiconductorindustry can be used as known in the art of photo-lithography.

In various embodiments, the substrate can be prepared from two or morelaminated layers. The term “detection-compatible material” as usedherein refers to the optical detection with a substrate that includesone or more layers which provide an optical transparency for each samplechamber. By way of example, the optical transparency may permitdetection of a luminescent dye. Silica-based glasses, quartz,polycarbonate, or an optically transparent plastic layer may be used,for example. Selection of the particular detection-compatible materialdepends in part on the optical properties of the material and thedetection mechanism. For example, in luminescent dye-based assays, thematerial should have low fluorescence emission at the wavelength(s)being measured. The detection-compatible material should also exhibitminimal light absorption for the signal wavelengths of interest.However, in some cases, for example, to minimize cross-talk, it may bedesirable to provide a substrate material that has relatively highabsorption. Examples of such materials are described at U.S. Pub. No.2005/0226779A1.

In various embodiments, other layers in the substrate can be formedusing the same or different materials. The term “assay-compatiblematerial” as used herein refers to the interaction of assay reagents andassay conditions (heat, pressure, pH, etc.) with the substrate material(hydrophobic, hydrophilic, inert, etc.). For example, the layer orlayers, such as a film defining the sample chambers can be formedpredominantly from a material that has high heat conductivity, such assilicon, a heat-conducting metal, or carbon fill in plastic. The siliconsurfaces that contact the sample can be coated with an oxidation layeror other suitable coating, to render the surface more inert and make itan assay-compatible material. Similarly, where a heat-conducting metalis used in the substrate, the metal can be coated with anassay-compatible material, such as a plastic polymer, to preventcorrosion of the metal and to separate the metal surface from contactwith the sample. The suitability of a particular surface should beverified for the selected assay as known by the conditions and reagentsused in the assay.

In various embodiments, for optical detection, the opacity ortransparency of the substrate material defining the sample chambers, forexample, the base, can have an effect on the permissible detectorgeometries used for signal detection. For the following discussion,references to the “upper wall” of a detection chamber may refer to thechamber surface or wall through which the optical signal is detected,and references to the “lower wall” of a chamber may refer to the chambersurface or wall that is opposite the upper wall. For example, the upperwall can be formed by the base or the film, and the lower wall by theother, respectively.

In various embodiments, in fluorescence detection the substrate materialdefining the lower wall of the sample chambers can be optically opaque,and the sample chambers can be illuminated and optically scanned throughthe same surface (i.e., the top surfaces of the chambers which areoptically transparent). Thus, for fluorescence detection, the opaquelower wall material can exhibit low reflectance properties so thatreflection of the illuminating light back toward the detector can beminimized. Opacity also may prevent collection of background signalsfrom a thermal cycler block or other instrumentation used to test thesubstrates. In other cases, the substrate lower wall of the samplechambers may be reflective so that more fluorescent signal is collected.

In various embodiments, in fluorescence detection the substrate materialdefining the upper wall of the sample chambers can be optically clear,the chambers can be illuminated with excitation light through the sidesof the chambers (in the plane defined collectively by the samplechambers in the substrate), or more typically, diagonally from above(e.g., at a 45 degree angle), and emitted light is collected from abovethe chambers (i.e., through the upper walls, in a directionperpendicular to the plane defined by the detection chambers). The upperwall material can exhibit low dispersion of the illuminating light inorder to limit Rayleigh scattering.

In various embodiments, in fluorescence detection the substrate materialdefining the entirety of the substrate can be optically clear, or atleast the upper and lower walls of the chambers can be optically clear,the chambers can be illuminated through either wall (upper or lower),and the emitted or transmitted light is measured through either wall asappropriate. Illumination of the chambers from other directions can alsobe possible as already discussed above.

In various embodiments, in chemiluminescence detection, where light of adistinctive wavelength is typically generated without illumination ofthe sample by an outside light source, the absorptive and reflectiveproperties of the substrate can be less important, provided that thesubstrate provides at least one optically transparent window fordetecting the signal.

In various embodiments, the substrate can be designed to provide avacuum-tight environment within the sample-distribution network forsample loading, and also to provide sample chambers having carefullydefined reaction volumes. It is desirable to ensure that the network andassociate sample chambers do not leak. Accordingly, lamination ofsubstrate layers to one another can be accomplished so as to ensure thatall chambers and channels are well sealed.

In various embodiments, the substrate layers can be sealably bonded in anumber of ways. A suitable bonding substance, such as a glue orepoxy-type resin, can be applied to one or both opposing surfaces thatwill be bonded together. The bonding substance may be applied to theentirety of either surface, so that the bonding substance (after curing)can come into contact with the sample chambers and the distributionnetwork. In this case, the bonding substance is selected to becompatible with the sample and detection reagents used in the assay.Alternatively, the bonding substance can be applied around thedistribution network and detection chambers so that contact with thesample can be minimal or avoided entirely. The bonding substance mayalso be provided as part of an adhesive-backed tape or membrane, whichis then brought into contact with the opposing surface. In yet anotherapproach, the sealable bonding is accomplished using an adhesive gasketlayer, which is placed between the two substrate layers. In any of theseapproaches, bonding may be accomplished by any suitable method,including pressure-sealing, ultrasonic welding, and heat curing, forexample.

In various embodiments, a pressure-sensitive adhesive (PSA) can be usedin constructing the substrate. PSA films which can be applied to asurface and adhered to that surface are obtained by applying pressure tothe film. Normally, pressure is applied throughout the whole film, sothat the whole film can adhere to the surface. PSA films can havethreshold pressure in order to activate the adhesion. The thresholdpressure can be very low. By applying pressure to some selected regions,the bonding can be limited to those regions only, thus allowingobtaining a bonding pattern. This way channels and chambers can bedefined. The elastic properties of the film can then be used topressure-drive a fluid through the unbonded regions, since the filmwould deform under the liquid pressure, thus opening up a channel.Eventually, the channel could be sealed by applying pressure on theportion of the film defining the channel. PSA films can be eitherhydrophobic or hydrophilic. PSA films can have hydrophobic andhydrophilic areas on the same film to provide areas of different wettingcharacteristics, properly patterned, to provide, for example fluid flowin sample introduction channels and gas venting in venting channels.Thus, by providing differing regions of hydrophobicity andhydrophilicity of the films, control over fluid flow through a devicemay be achieved. In various embodiments, PSA films that are hydrophiliccan have the hydrophilic properties deteriorate in a matter of days. Thelack of stability (hydrophilic film turning into hydrophobic) canprovide controllable, irreversible or reversible, changes (upontemperature change, heat addition, UV exposure, or just time delay aftercuring) in the wetting nature of the film. In various embodiments, PSAfilms can have different porosities and permeabilities to a gas. Ahighly permeable PSA film can be more advantageous than alow-permeability one for instance to vent the sample chambers. Further,a PSA film whose permeability/porosity can be modified in a reversiblefashion with temperature change, and/or in an irreversible fashion byheat addition or UV exposure can be used for sample distribution andthen sealed for sample processing. In various embodiments, PSA films canbe hydrophilic, provide solvent resistance, maintain the adhesioncharacteristics at a high temperature (95-100 degree Celsius), and canbe optically clear with low auto-fluorescence. In various embodiments,PSA films can be thermally expandable to swell at desired locations andclose off channels.

In various embodiments, the substrate of the present teaching can beadapted to allow rapid heating and cooling of the sample chambers tofacilitate reaction of the sample with the analyte-detection reagents,including luminescent dyes. In one embodiment, the substrate can beheated or cooled using an external temperature-controller. Thetemperature-controller is adapted to heat/cool one or more surfaces ofthe substrate, or can be adapted to selectively heat the sample chambersthemselves. To facilitate heating or cooling with this embodiment, thesubstrate can be formed of a material that has high thermalconductivity, such as copper, aluminum, or silicon. Alternatively, basescan be formed from a material having moderate or low thermalconductivity, while the film can be formed form a conductive materialsuch that the temperature of the sample chambers can be convenientlycontrolled by heating or cooling the substrate through the film,regardless of the thermal conductivity of the base. For example, thefilm can be formed of an adhesive copper-backed tape.

In various embodiments, the sample chambers of the substrate can bepre-loaded with detection reagents that are specific for the selectedanalytes of interest. By way of example, such reagents may be depositedin the sample chambers in liquid form and dried (e.g., lyophilized). Forexample, reagents for nucleic acid analysis of a biological sample maybe preloaded in the substrate, for example, in the sample chambers. Insuch embodiments, the substrate may then be loaded with sample whenbiological testing is desired to be performed by supplying sample to thesubstrate containing the pre-loaded reagent(s). The detection reagentscan be designed to produce an optically detectable signal via any of theoptical methods known in the field of detection. It will be appreciatedthat although the reagents in each detection chamber can containsubstances specific for the analyte(s) to be detected in the particularchamber, other reagents for production of the optical signal fordetection can be added to the sample prior to loading, or may be placedat locations elsewhere in the network for mixing with the sample.Examples of such reactions are described in U.S. Pub. No.2005/0260640A1. Whether particular assay components are included in thedetection chambers or elsewhere will depend on the nature of theparticular assay, and on whether a given component is stable to drying.Pre-loaded reagents added in the detection chambers during manufactureof the substrate can enhance assay uniformity and minimize the assaysteps conducted by the end-user.

In various embodiments, the analyte to be detected may be any substancewhose presence, absence, or amount is desirable to be determined. Thedetection means can include any reagent or combination of reagentssuitable to detect or measure the analyte(s) of interest. It will beappreciated that more than one analyte can be tested for in a singledetection chamber, if desired.

In one embodiment, the analytes are selected-sequence polynucleotides,such as DNA or cDNA, RNA, and the analyte-specific reagents includesequence-selective reagents for detecting the polynucleotides. Thesequence-selective reagents include at least one binding polymer that iseffective to selectively bind to a target polynucleotide having adefined sequence. The binding polymer can be a conventionalpolynucleotide, such as DNA or RNA, or any suitable analog thereof,which has the requisite sequence selectivity. Other examples of bindingpolymers known generally as peptide nucleic acids may also be used. Thebinding polymers can be designed for sequence specific binding to asingle-stranded target molecule through Watson-Crick base pairing, orsequence-specific binding to a double-stranded target polynucleotidethrough Hoogstein binding sites in the major groove of duplex nucleicacid. A variety of other suitable polynucleotide analogs are also knownin the art of nucleic acid amplification. The binding polymers fordetecting polynucleotides are typically 10-30 nucleotides in length,with the exact length depending on the requirements of the assay,although longer or shorter lengths are also contemplated.

The present teachings can find utility in a wide variety ofamplification methods, such as PCR, Reverse Transcription PCR (RT-PCR),Ligation Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification(NASBA), self-sustained sequence replication (3SR), strand displacementactivation (SDA), Q (3replicase) system, isothermal amplificationmethods, and other known amplification method or combinations thereof.Additionally, the present teachings can find utility for use in a widevariety of analytical techniques, such as ELISA; DNA and RNAhybridizations; antibody titer determinations; gene expression;recombinant DNA techniques; hormone and receptor binding analysis; andother known analytical techniques.

In one embodiment, the analyte-specific reagents include anoligonucleotide primer pair suitable for amplifying, by polymerase chainreaction, a target polynucleotide region of the selected analyte that isflanked by 3′-sequences complementary to the primer pair. In practicingthis embodiment, the primer pair is reacted with the targetpolynucleotide under hybridization conditions which favor annealing ofthe primers to complementary regions of opposite strands in the target.The reaction mixture is then thermal cycled through several, andtypically about 20-40, rounds of primer extension, denaturation, andprimer/target sequence annealing, according to well-known polymerasechain reaction (PCR) methods. Typically, both primers for each primerpair are pre-loaded in each of the respective sample chambers. Theprimer also may be loaded along with the standard nucleotidetriphosphates, or analogs thereof, for primer extension (e.g., ATP, CTP,GTP, and TTP), and any other appropriate reagents, such as MgCl₂ orMnCl₂. A thermally stable DNA polymerase, such as Taq, Vent, or thelike, may also be pre-loaded in the chambers, or may be mixed with thesample prior to sample loading. Other reagents may be included in thedetection chambers or elsewhere as appropriate. Alternatively, thedetection chambers may be loaded with one primer from each primer pair,and the other primer (e.g., a primer common to all of sample chambers)can be provided in the sample or elsewhere. If the targetpolynucleotides are single-stranded, such as single-stranded DNA, cDNA,or RNA, the sample is preferably pre-treated with a DNA- orRNA-polymerase prior to sample loading, to form double-strandedpolynucleotides for subsequent amplification. Also, a reversetranscription enzyme may be used to pretreat RNA to cDNA. Thispre-treatment can be provided in the cartridge.

In various embodiments, the presence and/or amount of targetpolynucleotide in a sample chamber, as indicated by successfulamplification, is detected by any suitable means. For example, amplifiedsequences can be detected in double-stranded form by including anintercalating or crosslinking dye, such as ethidium bromide, acridineorange, or an oxazole derivative, such as, Cyber Green, for example,which exhibits a fluorescence increase or decrease upon binding todouble-stranded nucleic acids. The level of amplification can also bemeasured by fluorescence detection using a fluorescently labeledoligonucleotide. In this embodiment, the detection reagents include asequence-selective primer pair as in the more general PCR method above,and in addition, a sequence-selective oligonucleotide (FQ-oligo)containing a fluorescer-quencher pair. The primers in the primer pairare complementary to 3′ regions in opposing strands of the targetanalyte segment which flank the region which is to be amplified. TheFQ-oligo is selected to be capable of hybridizing selectively to theanalyte segment in a region downstream of one of the primers and islocated within the region to be amplified. The fluorescer-quencher paircan include a fluorescent dye and a quencher which are spaced from eachother on the oligonucleotide so that the quencher is able tosignificantly quench light emitted by the fluorescer S at a selectedwavelength, while the quencher and fluorescer are both bound to theoligonucleotide. The FQ-oligo can include a 3′-phosphate or otherblocking group to prevent terminal extension of the 3′ end of the oligo.The fluorescer and quencher dyes may be selected from any dyecombination having the proper overlap of emission (for the fluorescer)and absorptive (for the quencher) wavelengths while also permittingenzymatic cleavage of the FQ-oligo by the polymerase when the oligo ishybridized to the target. Suitable dyes, such as rhodamine andfluorscein derivatives, and methods of attaching them, are well known inthe art of nucleic acid amplification.

In another embodiment, the detection reagents include first and secondoligonucleotides effective to bind selectively to adjacent, contiguousregions of a target sequence in the selected analyte, and which can beligated covalently by a ligase enzyme or by chemical means as known inthe art of oligonucleotide ligation assay, (OLA). In this approach, thetwo oligonucleotides (oligos) can be reacted with the targetpolynucleotide under conditions effective to ensure specifichybridization of the oligonucleotides to their target sequences. Whenthe oligonucleotides have base-paired with their target sequences, suchthat confronting end subunits in the oligos are base-paired withimmediately contiguous bases in the target, the two oligos can be joinedby ligation, e.g., by treatment with ligase. After the ligation step,the detection wells are heated to dissociate unligated probes, and thepresence of a ligated, target-bound probe is detected by reaction withan intercalating dye or by other means. The oligos for OLA may also bedesigned so as to bring together a fluorescer-quencher pair, asdiscussed above, leading to a decrease in a fluorescence signal when theanalyte sequence is present. In the above OLA ligation method, theconcentration of a target region from an analyte polynucleotide can beincreased, if necessary, by amplification with repeated hybridizationand ligation steps. Simple additive amplification can be achieved usingthe analyte polynucleotide as a target and repeating denaturation,annealing, and ligation steps until a desired concentration of theligated product is achieved.

In another embodiment, the ligated product formed by hybridization andligation can be amplified by ligase chain reaction (LCR). In thisapproach, two sets of sequence-specific oligos are employed for eachtarget region of a double-stranded nucleic acid. One probe set includesfirst and second oligonucleotides designed for sequence-specific bindingto adjacent, contiguous regions of a target sequence in a first strandin the target. The second pair of oligonucleotides is effective to bind(hybridize) to adjacent, contiguous regions of the target sequence onthe opposite strand in the target. With continued cycles ofdenaturation, reannealing and ligation in the presence of the twocomplementary oligo sets, the target sequence is amplifiedexponentially, allowing small amounts of target to be detected and/oramplified.

In various embodiments, it will be appreciated that since the selectedanalytes in the sample can be tested for under substantially uniformtemperature and pressure conditions within the substrate, the detectionreagents in the various sample chambers should have substantially thesame reaction kinetics. This can be accomplished using oligonucleotidesand primers having similar or identical melting curves, which can bedetermined by empirical or experimental methods as are known in the art.In another embodiment, the analyte is an antigen, and theanalyte-specific reagents in each detection chamber include an antibodyspecific for a selected analyte-antigen. Detection may be byfluorescence detection, agglutination, or other homogeneous assayformat. As used herein, “antibody” is intended to refer to a monoclonalor polyclonal antibody, an Fc portion of an antibody, or any other kindof binding partner having an equivalent function. For fluorescencedetection, the antibody may be labeled with a fluorescent compound suchthat specific binding of the antibody to the analyte is effective toproduce a detectable increase or decrease in the compound'sfluorescence, to produce a detectable signal (non-competitive format).In an alternative embodiment (competitive format), the detection meansincludes (i) an unlabeled, analyte-specific antibody, and (ii) afluorescer-labeled ligand which is effective to compete with the analytefor specifically binding to the antibody. Binding of the ligand to theantibody is effective to increase or decrease the fluorescence signal ofthe attached fluorephore. Accordingly, the measured signal can depend onthe amount of ligand that is displaced by analyte from the sample. In arelated embodiment, when the analyte is an antibody, theanalyte-specific detection reagents include an antigen for reacting witha selected analyte antibody which may be present in the sample. Thereagents can be adapted for a competitive or non-competitive typeformat, analogous to the formats discussed above. Alternatively, theanalyte-specific reagents can include a mono- or polyvalent antigenhaving one or more copies of an epitope which is specifically bound bythe antibody-analyte, to promote an agglutination reaction whichprovides the detection signal.

In various embodiments, the selected analytes can be enzymes, and thedetection reagents include enzyme substrate molecules which are designedto react with specific analyte enzymes in the sample, based on thesubstrate specificities of the enzymes. Accordingly, detection chambersin the device may each contain a different substrate or substratecombination, for which the analyte enzyme(s) may be specific. Thisembodiment is useful for detecting or measuring one or more enzymeswhich may be present in the sample, or for probing the substratespecificity of a selected enzyme. Examples of detection reagents includechromogenic substrates such as NAD/NADH, FAD/FADH, and various otherreducing dyes, for example, useful for assaying hydrogenases, oxidases,and enzymes that generate products which can be assayed by hydrogenasesand oxidases. For esterase or hydrolase (e.g., glycosidase) detection,chromogenic moieties such as nitrophenol may be used, for example.

In various embodiments, the analytes are drug candidates, and thedetection reagents include a suitable drug target or an equivalentthereof, to test for binding of the drug candidate to the target. Itwill be appreciated that this concept can be generalized to encompassscreening for substances that interact with or bind to one or moreselected target substances. For example, the assay device can be used totest for agonists or antagonists of a selected receptor protein, such asthe acetylcholine receptor. In a further embodiment, the assay devicecan be used to screen for substrates, activators, or inhibitors of oneor more selected enzymes. The assay may also be adapted to measuredose-response curves for analytes binding to selected targets. Theassays also may be immunoassays.

Reference will now be made to various exemplary embodiments, examples ofwhich are illustrated in the accompanying drawings. Wherever possible,the same reference numbers are used in the drawings and the descriptionto refer to the same or like parts.

In various embodiments, as illustrated in FIG. 1, the substrate 10 hasan array of features providing parallel processing of several samples.The substrate can have dimensions, for example, 127.0 millimeters by85.7 millimeters providing 384 sample chambers. FIG. 2A illustrates aportion of substrate 10 showing the features. The top view is throughthe film 20 shown in ghost lines. FIG. 2B illustrates the exploded viewshowing film 20 with vents 40 aligning with base 30 and gas-permeablemembranes 50. Sample chambers 80 form a regularly spaced array. Sampleintroduced in sample ports 60 flows to main channels 70 and from thereto sample introduction channels 110 into sample chambers 80. Each samplechamber 80 is connected to venting channel 100 which joins ventingchamber 90 with sample chamber 80. Venting chamber 90 containsgas-permeable membranes 50 and aligns with vents 40. The membranetypically has a burst pressure of greater than 6 psi. In variousembodiments, the film 20 can be a PSA film with laser or mechanicallypunched vent 40. A membrane layer can be bound to the PSA film 20 anddye cut portions of the membrane layer can be removed leavinggas-permeable membranes 50. The base 30 can be injection molded oretched. The PSA film 20 with gas-permeable membranes 50 attached canthen be aligned with the base 30 and laminated together. In variousembodiments, the substrate can be mated with a plate providing aplurality of contacts to provide uniform pressure across the substratewhere the contacts do not provide substantial thermal transfer betweenthe substrate and the plate relative to the thermal transfer at thesurface of the substrate opposite to the plate. The plate can havethrough holes to permit light to pass from the sample chambers to adetector for detection. The plate is described in U.S. Pat. Pub. No.2001/0029794.

Examples of suitable membranes include gas-permeable membranes and/orporous membranes. For example, suitable porous membranes may includeGortex® and other similar materials known in the art of micro-porousmembranes. Suitable gas permeable membranes may include, for example,PDMS membranes. The membrane materials also can be liquid impermeable toprevent a sponging effect of the liquid that can reduce the volume inthe sample chamber. Some examples of suitable porous membrane materialsare described in U.S. Pat. No. 5,589,350 and some examples of suitablegas-permeable membrane materials are described in U.S. Pat. Pub. No.2005/0164373 Titled “Diffusion-Aided Loading System for MicrofluidicDevices,” the entire disclosures of both of which are incorporated byreference herein.

In various embodiments, as illustrated in FIGS. 3A-3B, the sealing plate120 has a plurality of sealing protrusions 130 such that each protrusioncan align with a sample chamber. The sealing plate 120 can be a thermaltransfer die to isolate each of the sample chambers. The sealingprotrusions can have dimensions of about, for example, 2.5 millimeters.FIG. 4 illustrates the alignment with substrate 10. Sealing plate 120seals sample introduction channels 110 and venting channels 100 as shownin FIG. 5 by gaps 140.

FIGS. 3C and 3D show another sealing plate according to exemplaryembodiments of the teachings herein. FIG. 3C shows an isometricperspective view of the sealing plate 320 in alignment for sealing asubstrate 310. FIG. 3D shows a view of the surface of the sealing plate320 having the protrusions 330 thereon. The sealing plate 320 has aplurality of sealing protrusions 330 in the form of substantiallyarc-shaped pins. The sealing protrusions 330 are configured and arrangedon the plate 320 such that when the plate 320 is aligned with thesubstrate 310, the protrusions 330 intersect the sample introduction andoutlet (venting) channels 375 and 376 to seal the sample chambers 380.Rather than encircling the entire sample chambers 380, the sealingprotrusions 330 extend just enough to contact the area of the substrate310 around the sample chambers 380 in the regions of the channels 375and 376 to perform the sealing function. Because the protrusions 330contact relatively small, focused regions of the substrate 310 duringsealing, less force on the sealing plate 320 may be required.

FIGS. 29 and 30 depict an exemplary embodiment of another instrumentthat may be useful for sealing the sample chambers 2980 of a substrate2910 (e.g., staking the substrate) and may be used instead of a sealingplate like that shown in FIGS. 3A-3B. The sealing instrument embodimentof FIGS. 29 and 30 includes a roller 2920 provided with a plurality ofsealing protrusions in the form of circumferential disks 2930 spacedfrom each other along the longitudinal axis of the roller 2920. Sealingof the substrate chambers 2980, for example, in a manner similar to thatshown in FIG. 5 by the gaps 140, may occur by rolling the disks 2930across the substrate 2910 using a sufficient force. For example, theforce may be enough to force an adhesive (e.g., a PSA) into the inletand outlet channels 2975 and 2976 and/or otherwise deform the channels2975 and 2976 at the locations that the disks 2930 cross over thechannels 2975 and 2976 to prevent flow communication between thechannels 2975, 2976 and the sample chambers 2980.

The number and positioning (spacing) of the disks 2930 may be selectedsuch that the roller 2920 may pass over the substrate 2910 once toisolate all of the sample chambers 2980, sealing both inlet and outletchannels 2975 and 2976 simultaneously. The number and positioning of thedisks 2930 may thus depend on a variety of factors, including but notlimited to, for example, the number of sample chambers, the arrangementof the sample chambers, and the arrangement of the inlet channels andoutlet channels. According to various exemplary embodiments, the numberof disks may be reduced by staking the shared main fluid channels 2970and 2971 between two lines of chambers 2980 in the embodiment of FIGS.29 and 30 instead of the inlet channels 2975 of each of the chambers2980. In various embodiments, it may be desirable to provide a mainfluid channel (or main fluid channels) with a zig-zag configuration sothat it can be intersected and sealed (staked) multiple times, forexample, with one pass of the roller 2920 over the substrate 2910.Further, in the case of shared outlet (e.g., venting) channels betweenadjacent rows of chambers, the number of disks also may be reduced.

With reference now to FIG. 31, another exemplary embodiment of a sealinginstrument in the form of a roller 3120 is depicted. In the embodimentof FIG. 31, the roller 3120 includes a plurality of sealing protrusionsin the form of circular pins 3130 provided around the outercircumference of the roller 3120. The pins 3120 may be aligned over arow of chambers 3180 in a substrate 3110 and seal the chambers 3180 in amanner similar to that depicted by the gaps 140 in FIG. 5. However,rather than sealing all of the chambers 3180 of the substrate 3110 atthe same time, like the sealing plate of FIGS. 3A and 3B, a few chambers3180 get sealed as the roller 3120 passes over the substrate 3110. Thisrequires less force to seal the chambers, since not all of the chambers3180 are sealed at once. Although each chamber 3180 in a row of chambers3180 (e.g., x-direction in FIG. 31) may be sealed simultaneously, it isalso envisioned that the roller 3120 may be moved such that less thanall chambers in a row are sealed at the same time as the roller 3120passes over the substrate 3110. For example, the roller 3120 of FIG. 31may be aligned over a portion of the substrate 3110 (e.g., shifted inthe x direction shown in FIG. 31) so as to seal only some chambers 3180in each row sealed with a pass of the roller 3120. The roller may thenbe shifted again over another portion of the substrate 3110 and passedover the substrate 3110 again to seal the remaining chambers 3180 ineach row.

It should be understood that the protrusions provided on the rollers inthe embodiments of FIGS. 29-31 may have a variety of different sizes,shapes, and arrangements and those of ordinary skill would understandhow to make rollers with sealing protrusions of other configurations andarrangements to perform desired sealing of the sample chambers of asubstrate. By way of example and not limitation, it is envisioned thatthe sealing protrusions 3130 of FIG. 31 may be replaced with the sealingprotrusions 330 of FIG. 3C, with the arrangement of the protrusions 330on the roller being selected so as to seal the sample introduction andventing channels from flow communication with the sample chambers of asubstrate.

In the exemplary embodiments of FIGS. 3A-3D and 29-31, the force on thesealing plates 120 and 320 and rollers 2920 and 3120 required to effectsealing of the substrate 10, 310, and 2910 and 3110 may be relativelylow, for example, due to the relatively small contact area between theroller and the substrate during sealing. Further, to reduce the forceapplied to achieve sealing, plural plates 120 and 320 or rollers 2910and 3110 may be used, with less sealing protrusions provided on each ofthe plural rollers or plates. In such a case, the positioning and/orshape of the protrusions on each of the plural rollers or plates used toperform complete sealing of all of the chambers of the substrate may beselected so as to achieve sealing of some portions of the substrate witha first roller or plate and other portions of the substrate with asecond roller or plate, etc. By way of example, the sealing protrusionson a first roller or plate may seal the outlet (e.g., venting channels)leading from the sample chambers, while the sealing protrusions on asecond roller or plate may seal the inlet channels leading to the samplechambers. Those having ordinary skill in the art would understand avariety of numbers and configurations of rollers and/or plates andsealing protrusions on those rollers and/or plates to accomplish desiredsealing of the substrate with a desired force applied.

In addition, it should be understood that in the embodiments of FIGS.29-31, the rolling of the rollers 2920 and 3120 over the substrates 2910and 3110 is intended to refer to relative motion between the rollers2920 and 3120 and the substrates 2910 and 3310. Thus, either the rollers2920 and 3120 can move while the substrates 2910 and 3110 remainstationary, vice versa, or both the rollers 2920 and 3120 and thesubstrates 2910 and 3110 may move. In another exemplary aspect, therollers 2920 and 3120 may be idle or can drive the motion of thesubstrates 2910 and 3110. Likewise, in the embodiments of FIGS. 3A-3Dand 4, the movement of the plates 120 and 320 and the substrates 10 and310 is relative.

It also is envisioned that, in the case of an integrated instrumentwhere the substrate passes through various stations, the sealing rollersor plates of the may be placed in the transfer path of the substrate.For example, the sealing rollers or plates may be placed at a locationafter the substrate has been filled (e.g., at a filling station) andbefore the next station, such as, for example, a thermocycling block orinstrument delivery port.

The exemplary sealing roller embodiments of FIGS. 29-31 may berelatively easy to manufacture. They also may facilitate appropriatealignment of the roller with the substrate during sealing, as alignmentis necessary only along the longitudinal axis of the roller and notalong the longitudinal axis of the substrate.

In various embodiments, sample preparation instrument 150 can take rawbiological sample from syringe 170 and prepare the sample forintroduction into substrate 10. Preparation of sample can includeextraction of nucleic acids and pre-treatment for detection as describedabove. The instrument 150 docks with several cartridges 160 that providepreparation. FIG. 7 illustrates the cartridge 160. Sample syringe inlet190 introduces the raw biological sample into the cartridge. Pre-filledreagent reservoirs 180 provide the analyte-specific reagents for theassay to be performed on substrate 10. The back of cartridge housing 200is open to permit interconnection of a flex circuit PCB device 210 withinstrument 150.

Various other exemplary embodiments may provide mechanisms for sealing,venting, controlling pressure, sample preparation, mixing, and/or otherfeatures useful in multiple analyte detection in a substrate inaccordance with teachings of the disclosure and are described in furtherdetail below.

As discussed above, once the sample chambers of a substrate have beenfilled, it may be desirable to seal filled chambers from flowcommunication with each other and the various distribution channels.Such sealing may be desirable, for example, before various operations,such as, for example, PCR, may be performed, and to preventcross-contamination between wells. It also may be desirable to provide amechanism for sealing that is relatively easily performed by a user ofthe biological testing device. Further, it may be desirable to provide amechanism for sealing the substrate that does not require the use ofsensors, heaters, and/or other components that may be relativelydifficult and costly to implement.

Referring to FIGS. 8A-8C, an exemplary embodiment of a substrate 810 isdepicted. It should be understood that the substrate 810 depicted inFIGS. 8A-8C is schematic for purposes of simplifying the drawings. Thus,FIG. 8A shows only three sample chambers 880 having inlet channels 875and outlet (venting) channels 876 connected to main fluid channels 870.The arrows in FIG. 8A illustrate the direction of flow of sample forfilling the sample chambers 880. It should be understood that thesubstrate 810 could include an array of sample chambers 880 connected byfluid distribution channels and may also include venting chambers (notshown in FIG. 8A). By way of example, the substrate 810 may includefeatures similar to that shown in FIG. 1 or may have otherconfigurations in accordance with the teachings herein.

The substrate 810 may include a base 830, which may have a configurationlike the bases described above. The base 830 may be covered with anadhesive-backed film 820. In various embodiments, the adhesive-backedfilm 820 may be, for example, a PSA film. With reference to thecross-section of the substrate 810 shown in FIG. 8B taken through line8C-8C in FIG. 8A, prior to filling the substrate 810 with biologicalsample, the adhesive-backed film 820 may be loosely applied over thebase 830 such that the channels 875 and 876 and sample chambers 880 arein flow communication, thereby permitting sample to be injected into thechannels 875, 876, and 870 to fill the sample chambers 880. After thesample chambers 880 have been filled and it is desired to seal thechambers 880, pressure may be applied to the film 820 so as to cause theadhesive 822 of the adhesive-backed film 820 to be forced into thechannels 870, 875 and 876 and partially into the chambers 880, asdepicted in FIG. 8C. Forcing the adhesive into the channels 870, 875,and 876 closes the channels 890, thereby preventing flow communicationbetween the chambers 880 and between the channels 875, 876, and 870, andchambers 880.

By way of example, in the case of a consumable product, theadhesive-backed film 820 may be loosely applied during manufacturing anda user of the substrate 810 may apply the pressure required for sealingafter loading the substrate with sample. Various mechanisms may be usedto apply pressure, for example, a substantially uniform pressure, oversubstantially the entire adhesive-backed film. For example, the pressurecould be applied by the user's hand pressing on the film 820. Othertechniques for applying the pressure include using a motorized steppingplate 825 (as schematically depicted in FIG. 9A) or a motorized roller826 (as schematically depicted in FIG. 9B). The plate 825 and/ormotorized roller 826 may be provided as part of separate instrumentationor as a separate mechanism to be used with the substrates, and may beused to seal numerous substrates. Those having ordinary skill in the artwould understand various mechanisms that may be used to apply asufficient force across the film layer 820.

According to various exemplary embodiments, in addition or as analternative to providing pressure to force the adhesive 822 to fill thechannels 875, 876, and 870, heat also may be used to facilitate thefilling of the channels with the adhesive 822. However, it is envisionedthat the use of heat is not necessary. The appropriate thickness of theadhesive layer 822 may be selected so as to perform adequate channelclosing without injecting too much adhesive into the chambers 880. Byway of example, the thickness of the adhesive layer 822 may be such thatthe entire depths of the channels are filled with adhesive, leaving noair pockets within the channels. Also, if sample is displaced duringsealing, it may be desirable to provide an adhesive thickness thatresults in a substantially consistent amount of fluid being displaced,while also minimizing the amount of wasted sample due to displacement.

FIGS. 10A-10C illustrate another exemplary embodiment that uses theadhesive of an adhesive-backed film to seal the channels from thechambers in a biological testing device. With reference to FIG. 10A, aprotruding portion 850 is provided in a portion of each of the inlet andoutlet channels 875 and 876 that lead to and from the chambers 880. Asshown in the cross-sectional view of FIG. 10B, the adhesive-backed film820 may be lightly applied prior to filling the substrate 810 with thesample, such that the channels 875, 876 and 870 may be in flowcommunication with the chambers 880, as described above with referenceto FIG. 8B. Once the substrate 810 has been filled with fluid (e.g.,biological sample) as desired, a pressure may be applied, for example,substantially uniformly over substantially the entire surface of thefilm 820. The pressure may be applied via any mechanism, including thosedescribed above with reference to the embodiments of FIGS. 8 and 9, and,optionally, heat may be applied. As a result of the pressure on the film820, the adhesive 822 of the film 820 will be forced into the channels875, 876 and 870. However, due to the presence of the protrudingportions 850, the adhesive 822 may come into contact with the protrudingportions 850 to seal the channels 875 and 876 from flow communicationwith the chamber 880 without filling the entire channels 875 and 876.The adhesive 822 may make substantially uniform contact oversubstantially the entire surface of the protruding portion 850, as shownin FIG. 100. Due to the adhesive 822 making contact with the protrudingportion 850 and not filling the entire channel depth, sealing may beimplemented with much of the sample remaining in the channels 875 and876 since a relatively small amount of sample will may be displaced dueto the entry of the adhesive throughout the channels 875 and 876. Forexample, the relatively small amount of sample that is displaced may beso as to slightly deform the film layer 820. Further, sealing may berelatively easy to accomplish since the adhesive layer may only need tocontact the protruding portions 850, rather than filling the entirechannels 875 and 876.

According to various exemplary embodiments, the protruding portions 850may be made of the same material as the base 830 and may be formed viainjection molding of the base 830. Other materials and techniques forforming the protruding portions 850 also may be used and would beunderstood to those having ordinary skill in the art. By way of example,and not limitation, the protruding portions 850 may have a height equalto about one-half the depth of the channels 875 and 876, and may spanacross the width of the channels (e.g., in the left to right directionshown in FIG. 10A).

In various exemplary embodiments, for example, when using a motorizedroller 826 such as that depicted in FIG. 9B to apply pressure to theadhesive-backed film 820, the roller 826 may be oriented such that itslongitudinal axis (e.g., its axle) is nonparallel (for example,perpendicular) to the channels 875 and 876 in which the protrudingportions 850 are placed when performing sealing. With such a nonparallelorientation to a channel during sealing, the roller 826 may contact onlya relatively small part of the channels 875 and 876 at a time, therebydisplacing a relatively small amount of fluid (e.g., sample), if at all,from the channels. In an exemplary embodiment, the roller 826 may beplaced at a 45 degree angle to both axes of the substrate. Placing theroller 826 in a parallel orientation to a channels 875 and 876 duringsealing may cause a relatively large portion of fluid (e.g., sample) inthe channels 875 and 876 to be displaced therefrom and may potentiallyincrease the pressure to a sufficient amount so as to break the sealformed between the film 820 and the base 830.

In various embodiments, a stationary rotating cam may be used, forexample, instead of the motorized plate or roller of FIGS. 9A and 9B, toapply a pressure to the adhesive film layer of a substrate in order toeffect sealing of the sample chambers, for example, of a stationary lineof sample chambers. In conjunction with such a motorized rotating cam, amember that applies pressure to substantially the entire film layerprior to the sealing by the cam may be used. By applying pressure to thefilm layer, a small amount of sample in the sample chambers may moveinto adjacent channels (e.g., sample introduction (inlet) and venting(outlet) channels). As the rotating cam comes into contact with thesubstrate to perform the sealing function, the pressure on the filmlayer applied by the member may be removed at a rate substantiallyproportional to the application of the pressure exerted by the cam andthe volume of the channels that will be reduced due to the adhesiveentering the channels. This may allow for accommodation of increasedpressure in the sample chambers caused by the sealing operation andprovide a defined sample volume in each sample chamber, which may makethermocycling more efficient.

FIGS. 11, 12A, and 12B schematically depict a side cross-sectional viewof another exemplary embodiment of an instrument useful for sealingfluid sample chambers in a biological testing device. As shown, thedevice may include a substrate 1110 that includes a base 1130 and acover 1135 (e.g., a PSA film layer) for the base 1130 that togetherdefine a plurality of channels 1190 in flow communication with aplurality of sample chambers 1180 such that the channels 1190 candeliver sample fluid to and from the sample chambers 1180. For purposesof simplification, the schematic depiction in FIGS. 11, 12A, and 12Bshow only sample chambers 1180 and introduction and outlet channels 1190in flow communication with those chambers 1180. It should be understood,however, that the substrate 1110 may include venting chambers, mainfluid channels, sample introduction channels, venting channels, a fluidinlet port for supplying fluid to the substrate, etc., in accordancewith the teachings herein.

The biological testing device may further include a sealing carrier1120, having a plate-like structure, that includes a plurality ofstaking blades 1122 on a side of the carrier 1120 facing the substrate1110. Prior to filling the substrate 1110, the carrier 1120 may beseparated from the substrate 1110 via a temporary mechanical mechanism,such as, for example, a film hinge, or may be separate form thesubstrate 1110 with no connection. The carrier 1120 may be brought intocontact with the substrate 1110 when sealing is desired. In variousembodiments, the carrier 1120 and substrate 1110 may be provided withmating pins and holes or other fastening mechanisms that are configuredfor insertion in one direction but prevent separation in the oppositedirection. The carrier 1120 may be separated such that the blades 1122are above and at a distance from the upper surface (as shown in FIGS. 11and 12) of the substrate 1110. In this separated configuration, thechannels 1190 are in flow communication with the sample chambers 1180and a biological sample may be loaded into the substrate 1110 via asuitable inlet, as depicted by the arrow in FIG. 12A. It should also beunderstood that an inlet for fluid supply may be provided on the upperor lower surface of the substrate 1110 via a port (not shown) that is inflow communication with the channel 1190 toward the right hand side ofthe figures.

Once the sample chambers 1180 of the substrate 1110 have been filled, asshown in FIG. 12A, a force may be applied to the carrier 1120 and/or thesubstrate 1110 so as to move the carrier 1120 toward the substrate 1110(e.g., as shown by the arrows in FIG. 12B). The force may be applied viaa variety of mechanisms, including but not limited to, for example, amotorized plate, a motorized roller, a clamp, a user's hand, or othersuitable mechanisms. The force may be sufficient to bring the carrier1120 into contact with the substrate 1110 (for example, by breaking ordeforming the mechanical mechanism that initially separates the carrier1120 from the substrate 1110). With the carrier 1120 and substrate 1110in the contacting position, as shown in FIG. 12B, the staking blades1122 are driven into the substrate 1110 at a location of the channels1190 proximate the chambers 1180. In various embodiments, each channel1190 may be aligned with a differing blade 1122. In other embodiments, asingle blade 1122 may be aligned with a plurality of channels 1190, forexample, the blades 1122 in FIGS. 11. 12A and 12B may extend into thedrawing sheet to seal differing channels positioned along a directioninto the drawing sheet.

The blades 1122 may pierce, deform, or otherwise alter the structure ofthe substrate 1110 at the locations so as to prevent flow communicationbetween the chambers 1180 and between the channels 1190 and the chambers1180. By way of example, the blades 1122 may pierce through film layer1135 and enter the channels 1190 so as to block flow between thechannels 1190 and corresponding chambers 1180. In addition to blockingflow communication between the channels 1190 and the chambers 1180, thecarrier 1120 may be configured to provide a seal (e.g., prevent flowcommunication) between the substrate 1110 and the exterior, for example,through the fill port in the substrate 1110.

The staking blades 1122 may be positioned relative to the carrier 1120such that they are properly aligned with the channels 1190 as desired toprevent flow communication between the channels 1190 and the samplechambers 1180 when the carrier 1120 is placed into the contactingposition with the substrate 1110 via the applied clamping force.Providing the blades 1122 as part of the carrier 1120 (e.g., an integralpart of the carrier 1120) may facilitate manufacturing and alignment ofthe blades 1122, as the appropriate alignment can be assured prior tosealing the substrate 1110 with the carrier 1120. The appropriatealignment of the blades 1122 with the channels 1190 may ensure reliablesealing of the substrate 1110 and may permit the use of relatively smallstaking blades 1122. Relatively small staking blades in turn may requireless force to drive the blades into the substrate 1110, for example, ascompared to larger staking blades. The shape, size, and material of theblades may be selected based on the thickness, material, and otherproperties of the cover 1135.

The carrier 1120 also may include optical apertures 1123, for example,windows, that are in substantial alignment with the sample chambers 1180when the carrier 1120 is in the sealing position, as depicted in FIG.12B. The optical apertures 1123 may thus permit optical detection of thesample chambers 1180 during biological testing/analysis. Other sealingplates according to embodiments of the teachings herein also may includesuch apertures.

In accordance with various exemplary embodiments, when using the deviceof FIGS. 11, 12A, and 12B to perform PCR, the substrate 1110 and carrier1120 may be placed between a clamp and a thermal block of a PCRinstrument. When the instrument applies a clamping force via the clampto the carrier 1120, for example, after the substrate 1110 has beenfilled with sample as desired, the carrier 1120 may move toward thesubstrate 1110. As the carrier 1120 moves toward the substrate, anymechanical mechanism that separates the carrier 1120 and the substrate1110 may fail (e.g., break or deform) such that the carrier 1120 movesinto a contacting position with the substrate 1110 and the blades 1122are driven into the substrate 1110, as depicted in FIG. 12B. The devicemay be placed between the clamp and thermal block of the PCR instrumenteither prior to or after filling of the substrate 1110 with sample,however, the clamping force will be applied after filling. It also maybe possible to use a clamping device that applies force to one or moresections of the substrate and/or carrier 1120 at a time to reduce theforce required for the blades 1122 to penetrate and seal the substrate1110. Further, in various embodiments, as suggested above, retainingclips or other mechanical connection means may be provided to secure thecarrier to the substrate in addition to the blades 1122 themselvesholding the carrier 1120 and substrate 1110 together.

In various embodiments, for example, in the case of PCR, the sealing ofthe substrate sample chambers may be implemented using the thermal blockthat is placed in contact with the substrate to perform thermocycling.Using the thermal block to perform the sealing function reduces a stepin the processing of the substrate, allowing the step of thermocyclingand sealing to be performed at the same time. Further, as will beexplained, using the thermal block to perform the sealing function mayenhance thermal contact and heat transfer between the thermal block andthe sample chambers, which may thereby reduce thermocycling times.

FIGS. 32 and 33 show schematic cross-sectional views of a substrate 3210including a base 3230 and film layer 3220. The base 3230 may define aplurality of features that, together with the film layer 3220 form afluid distribution network of fluid distribution channels and chambers,as discussed herein. In the view of FIGS. 32 and 33, for ease ofillustration, a single sample chamber 3280 is illustrated with channels3275 and 3276 leading to and from the chamber 3280. The substrate 3210may be made of a variety of materials in accordance with the teachingsherein.

To perform the sealing function, the thermal block 3250 is provided witha plurality of sealing protrusions (e.g., bumps) 3260 (which may be inthe form of an array), only one of which is depicted in FIGS. 32 and 33,that are configured and arranged to align with the sample chambers 3280.To perform thermal cycling, the thermal block 3250 is brought intocontact with the film layer 3220 of the substrate 3210 and force isapplied to move the substrate 3210 and thermal block 3250 together, forexample, via an optical detection mechanism acting on the side ofsubstrate 3210 opposite to the side the thermal block 3250 is in contactwith, as shown in FIG. 33. The protrusions 3260 may be configured suchthat the protrusions 3260 deform the film layer 3220 and partially enterthe chamber 3280, such that the film layer 3220 contacts the innerperiphery of the opening of the chamber 3280, sealing the chamber 3280from the channels 3275 and 3276, as shown in FIG. 33. Thus, theprotrusions 3260 on the thermal block 3250 permit direct isolation ofthe sample in the sample chambers 3280, rather than sealing portions ofthe channels 3275 and 3276.

The sealing protrusions 3260 of the thermal block 3250 may have variousconfigurations. In an exemplary embodiment, the dimensions of theprotrusions 3260 should be such that sufficient contact is made to sealthe chambers 3280 from the channels 3275 and 3276. By way of example,the sealing protrusions 3260 may have a substantially circularconfiguration with a radius that is slightly larger than the radius ofthe chambers 3280. The top of the sealing protrusions 3260 may besubstantially flat, as shown by protrusions 3260 a and 3260 b in FIG.34, or may be rounded, as shown by protrusion 3260 c in FIG. 34.Likewise, the sides of the sealing protrusions 3260 may be rounded, asshown by protrusions 3260 a and 3260 c, or substantially flat, as shownby protrusion 3260 b. In the case of a sealing protrusion having a flattop and rounded sides, like 3260 a, a rounded protrusion may be formedwith its top cut off so as to be flat. A rounded configuration mayprovide a slightly larger alignment tolerance with the sample chambersby permitting a sliding movement to be implemented during positioningthe thermal block in contact with the substrate.

According to exemplary embodiments, the sealing protrusions 3260 may bemachined directly on the thermal block 3250 or may be formed on a metalinsert secured to the thermal block 3250.

Due to the sealing protrusions of a thermal block entering the samplechambers to perform sealing, as discussed above, it may be desirable topermit a relatively small amount of displaced sample contained in thechambers to escape. FIGS. 35 and 36 illustrate exemplary embodimentsthat permit the escape of a small amount of displaced sample from thesample chambers when the sealing protrusions of a thermal block enterthe sample chambers to seal the chambers.

Referring first to FIG. 35, a small recess 3261 (e.g., dimple) may beformed in the sealing protrusions 3260 of the thermal block 3250 inorder to fill with displaced fluid as a result of reduction of thesample chamber volume during sealing. In various exemplary embodiments,the recess 3261 may form a hole through the sealing protrusions 3260from one side to another to permit a small amount of fluid displacedfrom the chamber to escape if needed so as to avoid the potential forover-pressurization of the chamber and potential adhesion failure of thefilm layer to the base during thermal cycling. FIG. 35A depicts anotherexemplary shape of a sealing protrusion 3260 d on a thermal block 3250for performing sealing and also permitting displaced sample in a samplechamber to escape. The sealing protrusion 3260 d includes raisedperimeter portions 3263 and a recessed center portion 3264. The raisedperimeter portions 3263 may enter the sample chamber proximate the edge(periphery) of the sample chamber and seal the sample chamber. Therecessed center portion 3264 may permit any displaced sample from thechamber to escape during sealing.

FIG. 36 represents another exemplary embodiment that may be used toprotect against over-pressurization and/or adhesion failure when thethermal block sealing protrusions 3260 enter the chambers 3280. In FIG.36, a side escape channel 3278 that is slightly deeper than the feedchannels 3275 and 3276 may be provided in flow communication with eachsample chamber 3280. A main fluid channel 3270 that is used to supplysample to the inlet feed channel 3275 also is depicted in the exemplaryembodiment of FIG. 36. During filling, a small displaced amount ofsample may escape through the escape channel 3278, and may also leave asmall amount of air at the end of the escape channel 3278, which may endin a venting chamber 3279 similar to the venting chambers 3290 at theend of the vent (outlet) channel 3276, as are described herein. Byforming the escape channel 3278 with a greater depth than the channels3275 and 3276, when the sealing protrusions of the thermal block enterthe chamber to seal off the channels 3275 and 3276, for example, asdepicted in FIG. 33, the sealing protrusions 3260 may not fully seal thechannel 3278 due to its greater depth. This may permit any displacedfluid in the chamber 3280 to pass into the escape channel 3278 duringsealing and thermocycling, which in turn, may reduce the potential forover-pressurization in the chamber 3280 and adhesion failure (e.g.,leakage) of the film layer 3220.

Although the embodiments of FIGS. 32-36 above described a thermal blockhaving sealing protrusions (sealing protrusions 3260) that mate withsample chambers to perform sealing, it is envisioned that protrusions ona thermal block also may be configured and arranged to achieve a sealingpattern similar to that shown by the gaps 140 of FIG. 5. In other words,the thermal block and sealing protrusions thereon may contact and sealthe substrate at locations of the sample introduction and ventingchannels in flow communication with each sample chamber. Those ofordinary skill in the art would understand how to configure and arrangesealing protrusions on a thermal block to accomplish this type ofsealing.

FIGS. 38 and 39A-39C depict yet another exemplary approach for achievingsealing of sample chambers in a substrate for biological testing.Referring to FIG. 38, a schematic representation of a substrate 3910 isshown, showing only three sample chambers 3980 for ease of illustration.In accordance with the teachings herein, the substrate 3910 may comprisea base 3930 and a film layer covering the base to form the sampledistribution network shown. In the exemplary embodiment, each samplechamber 3980 is in flow communication with a main fluid supply channel3970 via a sample introduction (inlet) channel 3975. A venting channel3976 leads from each sample chamber 3980 to a venting chamber 3990. Athrough hole (not shown) leads through the substrate 3910 from eachventing chamber 3990 to a corresponding venting chamber 3992 provided ina main fluid outlet channel 3972 that connects to the main fluid supplychannel 3970, for example, in a U-shaped bend as shown. The main fluidoutlet channel 3972 terminates in an overfill chamber 3995. Adissolvable plug of material 3900 is positioned in the main fluidchannel 3970 just downstream of the last introduction channel 3975 andcorresponding chamber 3980 and upstream of the venting chamber 3992corresponding to that chamber 3980. For example, the dissolvable plug3900 may be positioned before the U-shaped junction of the main fluidsupply channel 3970 and the main fluid outlet channel 3972. The plug3900 may be made of a material that can fill the channel 3970 to blockfluid flow temporarily as the material dissolves at a controlled rate.By way of example, the plug 3900 may be made of polyethylene glycol.

FIGS. 39A-39C illustrate various exemplary steps to fill and seal thesubstrate 3910. In FIG. 39A, sample S (e.g., a biological sample) isintroduced to the substrate 3910. The sample S may be pumped through themain fluid supply channel 3970, the introduction channels 3975, and intothe sample chambers 3980 via pressure from a volume of oil (not shown inFIG. 39A), or other substance that is immiscible with the sample, thatis pumped behind the sample S. As shown in FIG. 39A, the sample S thatis introduced is sufficient to fill the sample chambers 3980, theintroduction and venting channels 3975 and 3976, the venting chambers3990, and the main fluid supply channel 3970 from the inlet of thesubstrate 3910 (at the right hand side in FIG. 39A) up to the plug 3900.The plug 3900 prevents the sample S from advancing past the plug 3900until the sample S has a chance to fill the various chambers andchannels, as shown in FIG. 39A.

Once the substrate 3910 has been filled with sample S, as depicted inFIG. 39A, the plug 3900 may begin to dissolve, thus allowing anyremaining supply of sample S to the substrate 3910 and the oil O behindit to flow in the main fluid supply channel 3970 past the location ofthe plug 3900, as shown in FIG. 39B. The oil O may continue to besupplied to the substrate 3910 such that it fills the main fluid supplychannel 3970, the main fluid outlet channel 3972, the venting chambers3992, and reaches the overfill chamber 3995, as shown in FIG. 39C. Dueto the immiscibility of the oil O and sample S, once the oil fills theportions of the substrate 3910 described above and shown in FIG. 39C,the oil acts to seal the inlet and outlet of each of the sample chambers3980, for example, so that further processing of the sample in thechambers 3980 may occur. The total volume of sample S and oil O that aresupplied to the substrate 3910 may be selected so as not to fill theoverfill chamber 3995 completely. According to various embodiments, themain outlet channel 3972 may have a volume that is larger than the mainfluid supply channel 3970 since the channel 3972 fills with oil and doesnot affect the waste ratio of the sample, assuming the time required topump the oil through the main fluid supply channel 3970 is not toogreat.

In an exemplary aspect, the pumping of the sample into the substrate3910 may be at a substantially constant pressure so that the pressure isnot excessive so as to burst the seals during the time between fillingthe last well and breaking through the plug 3900. To allow the sample Sto reach the plug 3900, a vent hole (not shown) that permits gas (e.g.,air) to escape the substrate 3910 may be provided. According to variousembodiments, a mechanical sealing mechanism may be desired at the inletand outlet of channels 3970 and 3972 to prevent oil from being pumpedout due to potentially expanding sample S, for example, during PCRand/or thermocycling. In various embodiments, it also may be desirableto exert pressure on the oil to pressurize the fluids to reduce bubbleformation. In an exemplary aspect, such force may be placed on the filmlayer covering the base.

According to various embodiments, the oil sealing approach describedabove may include variations. By way of example, and not limitation,instead of the dissolvable plug 3900, a burst valve or a Timavo valvecan be used. In this case, rather than waiting for the plug to dissolve,the sample slug can be immediately followed with an oil slug. Also,rather than utilizing the vents described above, membranes (porous orgas permeable) can be used between the venting channel and the mainfluid outlet channel. In yet another exemplary embodiment, in place ofthe vents described with reference to FIGS. 38 and 39A-39C, ahydrophobic stop can be used between the venting channel and the mainfluid channel. Further, the configuration of the main channels may bemore along the lines of FIG. 8A, where sample is introduced along onemain channel, and venting occurs along the other main channel, withoutthe two channels being connected in the U-shaped junction of theembodiment of FIGS. 38 and 39A-39C. The main venting channel couldeither terminate in a large overfill chamber or use a valve to a wasteport. The sample can be followed directly with oil in the main fluidsupply channel. After the sample has been filled, oil can be introducedinto the main venting channel, either at the same time as the oil isintroduced into the main fluid supply channel, or either one can precedethe other. This configuration can be combined with any of the ventingapproaches in accordance with the teachings herein.

FIGS. 40A-40C depict yet another exemplary embodiment of an approach toseal sample chambers in a substrate. The embodiment of the substrate4010 shown in the partial views of FIGS. 40A-40C is similar in design tothe substrate 3910 of FIGS. 38 and 39A-39C with respect to the mainfluid supply channel 4070, sample introduction channels 4075, samplechambers 4080, and main outlet channel 4072. In the embodiment of FIGS.40A-40C, the sample chambers 4080 are in flow communication with ventingchambers 4090 via venting channels 4076 that are tunneled through thebase 4030, as shown best in FIGS. 40B and 40C. The venting chambers 4090are in flow communication with the main fluid outlet channel 4072 viaconnection channels 4073, as shown in FIGS. 40A-40C, which may be formedat the upper surface of the base 4030, rather than through the base 4030like channels 4076. For simplicity, FIGS. 40A-40C show only a partialview of the substrate 4010 depicting one sample chamber 4080. It shouldbe understood, however, that an array of such sample chambers 4080 andcorresponding introduction and venting channels and chambers areprovided.

The exemplary embodiment of FIGS. 40A-40C includes a plug (e.g., bead)4000 of super-absorbent material disposed in the venting chambers 4090.Such a super-absorbent material may be configured so as to absorb manytimes the bead's volume in water relatively rapidly and retain the waterunder relatively high pressure so that water is prevented from filteringthrough the bead and exiting therefrom. Examples of such super-absorbentmaterials that may be used to form the bead 4000 include, but are notlimited to, polymers, such as, for example, cross-linked polyacrylate.

Prior to filling the substrate 4010 with sample, the bead 4000 may bepositioned within the venting chamber 4090 such that it does not occupythe entire volume of the venting chamber 4090, as depicted in FIG. 40B.After sample S is introduced into the substrate 4010 and fills thechambers 4080, as depicted in FIG. 40C, the sample S exits through theventing channels 4076 and into the venting chambers 4090 in contact withthe beads 4000, causing the beads 4000 to absorb the sample S and swell.The swelling of the beads 4000 in turn occupies the venting chambers4090 and blocks the venting channels 4076, thereby sealing the chambers4080 so that sample therein cannot escape and further processing, suchas PCR, may be performed. In various embodiments, the sampleintroduction channels 4075 may be sealed via a fluid that is immisciblewith the sample, such as, for example, oil, in a manner similar to thatdescribed with reference to FIGS. 38 and 39A-39C. Other sealingmechanisms in accordance with the present teachings also may be used toseal the sample introduction channels 4075 and would be understood bythose skilled in the art based on the present teachings.

The beads 4000 may be configured so as not to block the connectionchannels 4073 when they have absorbed the sample S, thereby permittingescape of gas (e.g., air) into the main outlet (vent) channel 4072. Invarious embodiments, it may not be necessary to permit gas to escape theventing chambers and out of the substrate, however, since even atelevated temperatures, the super-absorbent beads 4000 may retain waterwithout the tendency for the water to evaporate. This may be especiallytrue when the beads are used for a relatively small fraction of theirabsorptive capacity.

In various embodiments, the beads 4000 may be substantially sphericaland have a diameter of about 1 mm prior to absorbing sample. It isenvisioned, however, that other shapes and sizes of the beads 4000 maybe used. In particular, the shape and size of the beads 4000, as well asthe configuration of the venting chamber 4090, may be selected such thatthe beads may swell and deform to substantially match the surface of theopening of the venting channel 4076 to the venting chamber 4090.Further, venting chamber 4090 may be small enough so that the bead 4000may only expand to a limited extent to prevent the bead 4000 fromabsorbing more than a predetermined amount of sample. In the exemplaryembodiment of FIGS. 40A-40C, the venting chamber 4090 may have asubstantially egg-shaped configuration, narrowing toward the end wherethe venting channel 4076 enters the chamber 4090.

According to various other embodiments, the venting channel 4076 may beprovided in the surface of the base 4030, as previously describedherein, rather than having the cylindrical configuration shown in FIGS.40A-40C. With such a configuration, it may be more difficult to ensurethat the beads 4000 expand so as to conform to the relatively squareprofile defined by the film layer covering the base. However, it may bepossible for the expansion of the beads 4000 to cause enough pressure onthe film layer 4020 to create a rounded top surface. Further, thesealing may not need to be complete if a secondary sealing mechanismalso is employed, such as, for example, the oil sealing described withreference to FIGS. 38 and 39A-39C.

In yet further exemplary embodiments, the bead 4000 may be in the formof a superporous hydrogel bead that acts to absorb sample and permitpassage of gas.

FIGS. 41A and 41B show an embodiment of a substrate 4110 that includesthe use of a porous, hydrophobic pellet 4100 inserted into a ventingchamber 4190 for both sealing the sample chambers 4180 and alsopermitting venting of gas from the substrate. Such a pellet 4100 may berelatively easy to manipulate and insert into the individual ventingchambers 4190. Further, placing the pellets 4100 substantially in thesame plane as the chambers 4180, as described below, may be advantageousduring thermocycling, for example, to provide more efficient heattransfer and/or a more effective thermal contact between a thermal blockand the substrate.

The substrate 4110 may have a configuration similar to that described inthe embodiment of FIGS. 40A-40C, with the exception that the ventingchannel 4176 is not formed through the substrate 4110, though it may beif desired, but rather on the surface of the substrate 4110. In theembodiment of FIGS. 41A-41B, the venting chamber 4190 may have asubstantially square edge at the side of the chamber 4190 proximate theventing channel 4176 and a tapered edge at the side proximate theconnection channel 4173. A substantially cylindrical porous hydrophobicpellet 4100 may be inserted into the venting chamber 4190 into contactwith the tapered side first and then pushed forward against the squareside, as depicted in FIG. 41B. The top surface of the pellet 4100 maysit slightly above the surface of the base 4130, as shown in FIG. 41B,and may be pushed down so as to be substantially flush with the surfaceof the base 4130 when the film layer 4120 is adhered to the base 4130.

After filling the substrate 4110 with sample S, the pellet 4100 mayprevent the sample from flowing past it, as shown in FIG. 41B, but couldallow for the passage of air due to its porous nature. According tovarious embodiments, the pellets 4100 for each venting chamber 4190 maybe formed from a coil of material, similar to a coil of string, and cutinto small pieces and placed in a consistent orientation so as to beproperly positioned in each venting chamber 4190.

In various embodiments, the sample introduction channels 4175 leading tothe sample chambers 4180 may be sealed via a fluid that is immisciblewith the sample, such as, for example, oil, in a manner similar to thatdescribed with reference to FIGS. 38 and 39A-39C. Other sealingmechanisms in accordance with the present teachings also may be used toseal the sample introduction channels 4175 and would be understood bythose skilled in the art based on the present teachings.

According to still further embodiments, a material capable of breakingdown to a gas, for example, with elevated temperatures may be used toseal sample chambers of a substrate. With reference to FIG. 42A, apartial perspective view of a substrate 4210 is depicted. The view inFIG. 42A shows a main fluid supply channel 4270 that is in flowcommunication with two sample introduction channels 4275 that lead tosample chambers (not shown). A material 4200 that is configured to breakdown into a gas at elevated temperatures is placed at the junctionbetween the main fluid supply channel 4270 and the introduction channels4275. According to various exemplary embodiments, the material 4200 maybe predeposited in the substrate 4210. The material 4200 may be or maybe made insoluble in water so it does not dissolve upon contact with thesample S as the sample S fills the substrate 4210, as depicted in FIG.42B. For example, the material may be deposited with an organic solvent.

The material 4200 may break down into a gas, like a blowing agent, atelevated temperatures, for example, at temperatures associated with PCRand/or thermocycling. By way of example, the material 4200 may turn togas at temperatures of about 90° C. Thus, as shown in FIG. 42C, thematerial 4200 may turn into a gas, for example, after the substrate 4210has been heated in the first step of a PCR process. This creates abubble B at the junction that prevents the sample S from migrating fromone sample chamber to another, thereby sealing the sample chambers.Although FIGS. 42A-42C depict the use of the material 4200 at thejunction between a main fluid supply channel 4270 and introductionchannels 4275, it should be understood that this approach also may beused to seal the sample chambers at their outlet (e.g., vent) sides. Invarious embodiments, the interior channel surfaces may be relativelyhydrophobic in order to prevent sample from wicking around the bubble B.

FIGS. 43A-43C depict yet another exemplary approach for sealing thesample chambers of a substrate for biological testing in accordance withthe present teachings. Again, for ease of discussion, only one samplechamber 4380 of the substrate 4310 is depicted in FIGS. 43A-43C. Withreference to FIG. 43A, the substrate 4310 includes a small blind chamber4382 in flow communication with the sample introduction channel 4375upstream of the sample chamber 4380 between the sample chamber 4380 anda main fluid supply channel 4370. Assuming that pressure filling is usedto supply sample to the substrate, the sample S progresses through themain fluid supply channel 4370, into the introduction channel 4375 andsample chamber 4380, and into the venting channel 4376, withoutsubstantially filling the blind chamber 4382, as shown in FIG. 43B. Thisis due to the pressure resistance of the relatively small blind chamber4382 in comparison to that of the chambers 4380. Thus, the blind chamber4382 contains trapped air after the remainder of the substrate has beenfilled.

Upon further processing of the sample in the sample chambers, forexample, during thermocycling and/or PCR, elevating the temperature ofthe substrate 4310 causes the trapped air in the chamber 4382 to expand,introducing an air pocket P in the portion of the introduction channel4375 slightly upstream and downstream of the chamber 4382, as shown inFIG. 43C. The air pocket P serves to seal the chamber 4380. Skilledartisans would understand that a blind chamber, similar to 4382, alsomay be provided on an outlet side of the sample chamber 4280 inconjunction with the venting channel 4376 to perform sealing.

Although FIGS. 43A-43C show the blind chamber 4382 being completelyfilled with trapped air, it should be understood that a small amount ofsample S may enter the blind chamber 4382 during filling. However, asthe substrate 4310 is heated during biological testing (e.g., PCR and/orthermocycling), the trapped air in the chamber 4382 will expands,forcing out any sample in the chamber 4382.

The embodiment of FIGS. 2A and 2B provides an exemplary configurationfor achieving venting of gas via membranes from a substrate of abiological testing device, while substantially preventing leakage of thesample and/or other fluids that fill the substrate. Various additionalexemplary embodiments for achieving venting in accordance with thedisclosure are described below.

With reference to FIG. 13, a partial perspective isometric view of anexemplary embodiment of a substrate 1310 including an array of featuresproviding parallel processing of several samples for carrying outbiological testing is illustrated. The substrate 1310 includes a base1330 and a film layer 1320. Sample chambers 1380 may form a regularlyspaced array, as depicted, for example, in FIG. 1. Sample introduced tothe substrate (e.g., via sample ports like sample ports 60 in theembodiment of FIG. 1 and not shown in FIG. 13) flows to main channel1370 and from there to sample introduction channels 1375 into samplechambers 1380. Each sample chamber 1380 is connected to a ventingchannel 1300 which joins a venting chamber 1390 with the sample chamber1380.

The base 1330 and film layer 1320 may be made from any of the materialsdescribed herein for the bases and film layers, respectively. By way ofexample, the film layer 1320 may be a COP film or a PSA film and may bethermally bonded to the base 1330, which may be manufactured from aplastic material, for example, such as COP. As described above withreference to the embodiment of FIGS. 2A and 2B, the film layer 1320 maybe provided with a plurality of vent holes 1340 configured to be alignedwith the venting chambers 1390 when the film layer 1320 is attached tothe base 1330. Rather than providing a die-cut membrane in each of theventing chambers 1390 like in the embodiment of FIGS. 2A and 2B,however, in the exemplary embodiment of FIG. 13, a venting membranestrip 1350 is provided on a side of the film layer 1320 that faces awayfrom the base 1330 (e.g., on the top surface of the film layer 1320).Providing such a strip configuration may facilitate manufacturing of thedevice, for example, by permitting a single strip to serve as the ventmembrane for a plurality of chambers 1390 and/or permitting relativelysimple manufacturing of the strip 1350 and manipulation of the strip1350 into position due to its relatively large size. The strip 1350 maybe attached to the film layer 1320 via adhesive and may be aligned witha row of venting chambers 1390, as depicted in FIG. 13. Thus, aplurality of membrane strips 1350 may be positioned on the top surfaceof the film layer 1320 so as to align with a plurality of rows ofventing chambers 1390 of the substrate 1310.

As shown in FIG. 13, according to various exemplary embodiments, theadhesive used to bond the membrane strip 1350 to the film layer 1320 mayalso be in the form of a strip 1325, for example, a PSA strip, providedon a bottom side of the membrane strip 1350 and having a length andwidth substantially similar to the membrane strip 1350. Vent holes 1326may be provided through the adhesive strip 1325 and in alignment withthe vent holes 1340 of the film layer 1320 and the venting chambers1390. The vent holes 1326 and the vent holes 1340 may be formed via alaser, mechanically punching, or other suitable technique for formingvent holes. If PSA strips 1325 are used to bond the membrane strips 1350to the film layer 1320, the film layer may be, for example, a COP filmlayer thermally bonded to the base 1330.

The membrane strips 1350 may be gas-permeable or porous and also liquidimpermeable so as to prevent leakages of the sample fluid from thesubstrate 1310. In various exemplary embodiments, the membrane stripsmay be made of materials such as those described above for the membranes40 of the embodiment of FIGS. 2A and 2B.

In various exemplary embodiments (not shown in the figures), instead ofproviding adhesive strips 1325 to bond the membrane strips 1350 to thefilm layer 1320, the film layer 1320 may be a double-sided adhesive PSAlayer such that adhesive on one side of the layer 1320 is used to bondthe film 1320 to the base 1330 and adhesive on the opposite side is usedto bond the membrane strips 1350 to the film layer 1320. In such anembodiment, the vent holes 1340 would be formed through the entire filmlayer 1320 including both adhesive sides of the layer 1320.

According to various exemplary embodiments, when using multi-chamberdevices for parallel processing of plural fluid samples, it may bedesirable to cycle the device through various temperatures. For example,it may be desirable to perform PCR, which requires thermal cycling ofthe device over a range of temperatures, for example from about 60° C.to about 95° C. In such cases, relatively precise temperature control inthe individual sample chambers of a substrate, as well as temperatureuniformity over the entire substrate area may be desired. Further, asdiscussed above, the ability to isolate the individual chambers afterfilling the chambers (e.g., to prevent flow communication between thechambers and between the chambers and channels that lead to and fromeach chamber, such as fluid introduction and venting channels), may bedesired in order to prevent cross-contamination during a biologicaltesting process such as PCR.

In order to perform thermal cycling, in accordance with variousexemplary embodiments, a thermal block may be placed in contact with themulti-chambered substrate. Typically, the thermal block is placed incontact with the film layer of the substrate that, together with thecavities formed in the base of the substrate form the fluid distributionnetwork made up of, for example, main fluid channels, a plurality ofsample chambers (e.g., in an array) in flow communication with the mainfluid channel by a plurality of sample introduction channels, and aplurality of venting chambers in flow communication with the pluralityof sample chambers via a plurality of venting channels. In other words,the thermal block may be positioned in contact with the substrate on theside of the base of the substrate that defines the various channel andchamber openings. For example, in the exemplary embodiments of FIGS. 1,2, and 13 the thermal block may be positioned in contact with the filmlayer 20 and with the membranes 1350 and film layer 1320.

Placing the thermal block on the same side of the base of the substratethat the membranes are located, however, may impair the ability toachieve effective and uniform thermal conductivity between the thermalblock and the sample chambers. In particular, the presence of membranes,whether disposed between the film layer and the base or on the side ofthe film layer facing away from the base, may cause an irregular surface(e.g., a “bumpy” surface). Such an irregular surface may prevent uniformcontact of the thermal block with the substrate, and in some cases, itmay be desirable to remove the membranes and add a metal laminationlayer instead of the film layer to perform PCR after the substrate hasbeen loaded with sample.

Further membranes in the form of strips of material may have a potentialto leak around the borders of the strips.

With reference to FIGS. 14-16, various exemplary embodiments areschematically illustrated that provide gas-permeable or porous membranesfor venting a substrate on a side of the substrate opposite to the sidethat is placed in contact with a thermal block for performing thermalcycling of the sample loaded into the substrate. FIG. 14 is an isometricperspective view of an exemplary embodiment of a substrate 1410 forwhich membrane strips 1450 are positioned on a side of the substrate1410 (the side facing up in FIG. 14) that is opposite to the side (theside facing down in FIG. 14) of the substrate 1410 that the thermalblock is placed in contact with during thermal cycling.

In the embodiment of FIG. 14, the substrate 1410 may comprise a base1430 covered with a film layer 1420 that together define a fluiddistribution network. FIG. 14 shows the side of the base 1430 lookingthrough the film layer 1420. As shown in FIG. 14, the base 1430 and filmlayer 1420 may together define a plurality of sample chambers 1480 thatform a regularly spaced array. Sample may be introduced in sample ports1460 and may flow to main fluid supply channels 1470 and from there tosample introduction channels 1475 into sample chambers 1480. Each samplechamber 1480 may be connected to a venting channel 1400 that joins aventing chamber 1490 with the sample chamber 1480. A venting throughhole (not shown) may be formed from each venting chamber 1490 andthrough the base 1430 so as to open at the side of the base 1430 facingupward in FIG. 14. Elements 1421, 1423 and 1431 are indexing holesprovided in each layer of the substrate 1410 to provide appropriatealignment of the substrate 1410 with other instrumentation, if needed,for example, during filling and/or sample analysis.

FIG. 28 shows an exemplary venting through hole 2800 that may be used inconjunction with a venting chamber 2890 associated with a sample chamber2880. The venting through hole 2800 in FIG. 28 may be used in the base1430 of FIGS. 14-16. As shown in the exemplary embodiment of FIG. 28,the through hole 2800 may have a conical shape with a smaller openingleading from the venting chamber 2890 and a larger opening formed at theunderside of the base. By way of example only, the opening leading fromthe venting chamber 2890 may be about 100 μm in diameter and the openingat the underside of the base may be about 500 μm in diameter. Theconical configuration shown in FIG. 28 is exemplary only, and ventingthrough holes in accordance with the teachings herein may have a varietyof configurations, including, for example, cylindrical. The shape andsize of the vent through holes may be selected based on various factors,including, for example, the manufacturing technique used to form thebase, desired venting, and other such factors.

With reference again to FIG. 14, the film layer 1420 and the base 1430may be made of any of the materials described herein for film layers andbases. In various embodiments, the film layer 1420 may be a metal orpolymer PSA film and may be bonded to the base 1430 via the adhesive,for example, by applying pressure and/or heat. Further, the base 1430may be etched, stamped, hot-embossed, or injection molded to form thevarious chambers and channels. Using a metal PSA film for film layer1420 may be desirable to achieve good thermal conductivity.

As shown in FIG. 14, the substrate 1410 may further include, on the sideof the base 1430 opposite to the side on which the film layer 1420 isplaced, a film layer 1425 formed with a plurality of vent holes 1426.The vent holes 1426 may be configured and arranged so as to besubstantially aligned with the vent through holes of the base 1430described above. The film layer 1425 may be made of any of the materialsdescribed herein as useful for forming a film layer. By way of example,the film layer 1425 may be a PSA film layer and may be configured to beadhesively bonded and aligned with the base 1430. In accordance withvarious exemplary embodiments, the vent holes 1426 may be formed in thefilm layer 1425 via laser or via mechanical punching. In an alternativeembodiment, the film layer 1425 may formed of a porous hydrophobicmaterial and the vent holes 1426 may be eliminated.

Gas-permeable or porous membrane strips 1450 may be placed in contactwith the film layer 1425 and in alignment with the vent holes 1426. Eachmembrane strip 1450 may be arranged and configured so as to cover a rowof vent holes 1426. Examples of porous membranes include Gortex® andother similar materials known in the art and examples of selectivelypermeable membrane materials include, for example, PDMS. The membranestrips 1450 can be liquid impermeable so as to prevent leakage of samplefrom the substrate and to prevent a sponging effect of the liquid thatcan reduce the volume in the sample chamber. Other suitable porousmembrane materials are described in U.S. Pat. No. 5,589,350 and othersuitable gas-permeable membrane materials are described in U.S. Pat.Pub. No. 2005/0164373 entitled “Diffusion-Aided Loading System forMicrofluidic Devices,” both of which are incorporated herein.

According to various embodiments, the film layer 1425 may be adouble-sided adhesive film layer, for example, a double-sided PSApolymer film, and the membrane strips 1450 may be adhered to the filmlayer 1425 via the adhesive provided on the side of the film layer 1425facing the strips 1450. In alternative exemplary embodiments (not shown)the film layer 1425 may be a PSA film having only one adhesive layerfacing the base 1430. The membrane strips 1450 may be adhered to theopposite side of the film layer 1425 via adhesive strips (e.g., PSAstrips) having substantially the same length and width as the membranestrips 1450. Thus, for example, the membrane strips 1450 may be adheredto the film layer 1425 via adhesive strips similar to adhesive strips1325 shown and described with reference to the embodiment of FIG. 13.Like the adhesive strips 1325 of FIG. 13, adhesive strips used to adherethe membrane strips 1450 to the film layer 1425 may be provided withvent holes that align with the vent holes 1426 of the film layer 1425and with the vent through holes (not shown) provided in the base 1430.When using PDMS membrane strips, additional adhesive may not be neededas PDMS is self-adhering.

With reference now to FIG. 15, another exemplary embodiment of asubstrate having membranes for venting positioned on a side of thesubstrate opposite to the side of the sample chamber and channelopenings in the base is shown. The exemplary embodiment of FIG. 15includes components and materials similar to those described above withreference to the exemplary embodiment of FIG. 14, with the referencelabels of such components being the same as those used in FIG. 14. Inaddition, in the exemplary embodiment of FIG. 15, recesses 1530 areformed in the base 1430. The recesses 1530 have substantially the samedimensions as the membrane strips 1550 and are configured to receive themembrane strips 1550 and a film layer attached to the membrane strips1550 for bonding the membrane strips 1550 to the base 1430. The recesses1530 may facilitate proper alignment of the membrane strips 1550relative to the vent holes 1426 and vent through holes (not shown) ofthe base 1430 during placement of the strips 1550 on the substrate 1410.Also, providing recesses 1530 having a depth substantially equal to thethickness of the membrane strips 1550 may permit the membrane strips1550 to be positioned flush with the upper surface of the substrate1410. As such, a thermal block may be positioned in contact with theupper surface (e.g., the membrane side of the substrate 1410) and maymake substantially uniform thermal contact with the upper surface,thereby enhancing uniform thermal conductivity. It should be understood,however, that a thermal block also may be positioned in addition orinstead in contact with the bottom surface of the substrate 1410 (e.g.,in contact with the film layer 1420), as described with reference to theembodiment of FIG. 14.

As described above with reference to the exemplary embodiment of FIG.14, in various embodiments, the membranes 1550 of FIG. 15 may be bondedto the surface of a single sided adhesive layer 1425 via adhesive strips(not shown), for example, PSA adhesive strips, having substantially thesame length and width as the membranes 1550. Again, however, ifself-adhering PDMS strips are used, additional adhesive is not needed.Regardless of how the membrane strips 1550 are attached within therecesses 1530, the depth of the recesses 1530 may be selected so as toaccommodate both the thickness of the membrane strips 1550 and thethickness of an adhesive layer such that membrane strips 1550 aresubstantially flush with the top surface of the substrate 1410. In otherwords, the top surface of the substrate 1410 and the membrane strips1550 placed in position in the recesses should be substantially flat anduniform.

Yet another exemplary embodiment of a substrate for parallel processingof biological samples that utilizes a venting membrane on the backsideof the substrate is shown in FIG. 16. The exemplary embodiment of FIG.16 includes many of the same components and materials as described abovewith reference to the exemplary embodiment of FIG. 14, and illustratedcomponents that are the same as those in the exemplary embodiment ofFIG. 14 are indicated by the same reference labels. The exemplaryembodiment of FIG. 16 differs from that of FIG. 14, however, in that themembrane strips 1450 are replaced with a single venting membrane layer1650 configured and arranged to cover substantially the entire topsurface of the substrate 1410, as depicted in FIG. 16.

Providing a single membrane 1650 may facilitate positioning andattaching of the membrane 1650 to the substrate 1410, may reduce thenumber of components, and thus also may facilitate manufacturing. Themembrane 1650 also may include a plurality of optical apertures 1655configured and arranged to be substantially aligned such that the samplechambers 1480 (shown in FIG. 14) can be optically detected duringbiological testing. It should be noted that optical detection of thechambers 1480 can occur through the optical apertures 1655 by providinga transparent film layer 1425 and transparent base 1430. The opticalapertures 1655 may be substantially circular, although apertures alsomay have shapes other than circular.

According to various embodiments, in a manner similar to that describedwith reference to FIG. 15, the base 1430 of FIG. 16 may be provided witha single large recessed region (not shown in the view of FIG. 16)configured to receive the film layer 1425 and membrane 1650. This maypermit the membrane 1650 to lie flush with the upper surface of the base1430.

By providing the venting membranes 1450, 1550, and 1650 on the side ofthe substrate 1410 opposite to the side that is placed in contact withthe thermal block during thermal cycling, as shown in the exemplaryembodiments of FIGS. 14-16, it may be possible to achieve a more uniformand effective thermal conduction between the thermal block and thesubstrate 1410. Moreover, isolation of the sample chambers may befacilitated. For example, if staking and/or filling channels withadhesive is used to effect isolation of the sample chambers (e.g.,blocking flow communication between sample chambers and between samplechambers and channels), such techniques may be performed at the side ofthe substrate opposite to the side on which the membranes are placed.Thus, a lower force may be applied to deform and/or puncture the filmlayer 1420 than would be required to deform and/or puncture both a filmlayer and membranes. Alternatively, sealing could occur on the same sideas the venting membranes, especially in the embodiment of FIG. 16 ifpressure is applied at the apertures 1655. The various membraneembodiments of FIGS. 14-16 also may facilitate sealing of the samplechambers of the substrate via the thermal block itself, for example, asshown and described with reference to the exemplary embodiments of FIGS.32-36. The various membrane embodiments of FIGS. 14-16 also mayfacilitate manufacturing of the device as the membranes are relativelyeasily manipulated and installed. Moreover, the thermal conductivity maybe improved by using a metal film layer 1420 and reducing thickness ofthat layer, which may be placed in contact with the thermal block duringthermocycling. In some embodiments, where it may be desirable to heatthe substrate from both sides (e.g., place a thermal block in contactwith the membrane side and opposite side of the substrate), opticaldetection may occur via illumination from the edges of the substrate,though chambers may be restricted to locations around the perimeter ofthe substrate.

According to various embodiments, a heated cover used for processing(e.g., a thermal block in a thermocycler), if placed in contact with theventing side of the substrate in FIGS. 14-16, for example, may alsoinclude holes or porous areas that align with the vent holes and ventingchambers to permit gas to escape during loading of the substrate whilein place in a thermocycler.

In some circumstances, it may be desirable to eliminate ventingmembranes at each of the venting chambers. For example, by eliminatingthe need for such membranes, manufacturing may be facilitated and lesscostly since handling and assembly of the membranes is not needed.Further, precise alignment of the membranes will not be required and thechances of misalignment of a membrane and potential consequent leakageof sample may be avoided. Also, isolation (e.g., sealing) of the samplechambers of the substrate may be facilitated and improved due to areduction in force needed to deform and/or penetrate substrate layers toachieve isolation, as removal of the membranes may provide less layersto deform and/or penetrate. Finally, removal of such venting membranesmay improve thermal conductivity and thermal uniformity, for example,during PCR thermal cycling, due to the provision of a substantially flatsurface with which a thermal block may be placed in contact and/or adecrease in thickness of the layers of the substrate that a thermalblock must act on.

According to various embodiments, a multi-chambered substrate mayinclude a plurality of micro-sized vent holes in the film layer that,together with the base, forms the fluid distribution network (e.g.,sample chambers, main fluid channel, sample introduction channels,venting channels, and venting chambers) in the substrate. Themicro-sized vent holes may function both as capillary stops to preventleakage of sample from a filled substrate and as vents to release gasfrom the substrate.

A partial, isometric, perspective view of a multi-chambered substrate1710 that eliminates the need to provide a membrane over each ventingchamber is depicted in FIG. 17. The substrate 1710 includes a base 1730and a film layer 1720 that is adhered to the base 1730. The base 1730defines a plurality of features and, with the layer 1720 placed inposition over the base, defines a main fluid channel 1770 configured toreceive the sample supplied to the substrate 1710 and distribute thesample to a plurality of sample introduction channels 1775 that are inflow communication to in turn supply the sample to a plurality of samplechambers 1780. Each of the sample chambers 1780 is in flow communicationwith a venting chamber 1790 via a venting channel 1700.

The film layer 1720 is provided with a plurality of micro-sized ventholes 1742 configured and arranged to be aligned with the ventingchambers 1790 when the film layer 1720 is in position on the base 1730.By way of example only, the film layer 1720 may be a PSA film layer withadhesive on one side used to attach the film layer 1720 to the base1730. The film layer 1720 may be, for example, a PSA polymer film or aPSA metal film. The vent holes 1742 may be sized so as to allow gas toescape from the substrate 1710 while creating a fluidic stop thatprevents the sample within the substrate from leaking through the holes1742. For example, capillary forces may prevent the sample from passingthrough the holes 1742 and out of the substrate 1710. In variousexemplary embodiments, the vent holes 1742 may have a dimension (e.g., adiameter) ranging from about 1 μm to about 10 μm, for example, about 5μm. In some embodiments, areas surrounding the vent holes 1742 may besubstantially free of adhesive to prevent adhesive from flowing (e.g.,cold-flowing) into and reducing the diameter of the vent holes.

In some cases, it may also be desirable to provide venting at the end ofthe fill channel 1770. Thus, the exemplary embodiment of FIG. 17 alsoincludes a venting chamber 1795 and corresponding vent membrane 1798provided at the end of the main fill channel 1770. The membrane 1798 maybe contained in the venting chamber 1795 between the film layer 1720 andthe base 1730, for example, similar to the membranes 50 discussed withreference to the exemplary embodiment of FIGS. 2A and 2B. The membrane1798 thus may be sized and configured to substantially fill the ventingchannel 1795. The membrane 1798 may be made of any material describedherein as suitable for such porous or gas-permeable membranes. A venthole 1749, which may be formed in the same manner and may have a similarstructure as the vent holes 1742, may be provided in the film layer 1720in a position aligned with the membrane 1798 and venting chamber 1795.The venting chamber 1795 is relatively large compared to the ventingchambers 1790. Although the exemplary embodiment of FIG. 17 depicts theuse of the venting chamber 1795, the membrane 1798, and the vent hole1749, a substrate like that in FIG. 17 but that does not include thosefeatures is also considered as within the scope of the invention. Insuch a case, sufficient venting may be provided solely by the use ofvent holes 1742 corresponding to each venting chamber 1790.

Various techniques may be used to provide the micro-sized vent holes1742 in the film layer 1720. According to various embodiments, lasermicro-machining (e.g., drilling) may be used to form the holes 1742 inthe film layer 1740. For example, a laser micro-machining process may beused to drill holes through the film layer 1740 after it has beenattached to the base 1730, without penetrating the base 1730. Oneexemplary laser micro-machining process developed by Oxford Lasers, Inc.(Oxon, United Kingdom) uses an ultraviolet cold laser process capable ofdrilling holes having a dimension (e.g., diameter) ranging from about 5μm to about 10 μm in the film layer 1720. This process may form about 10holes to several hundred holes per second, for example about 16 holesper second, in the film layer 1720 after the film layer 1720 has beenbonded to the base 1730, without damage to the base 1730.

The laser drilling process can be applied to a variety of materials,including, but not limited to, for example, silicon, glass, metal,and/or polyimide. Examples of holes laser-drilled in various materialsusing Oxford Lasers, Inc. instruments are shown in FIGS. 18A-18C. Inparticular, FIG. 18A shows a laser-drilled hole of about 5 μm indiameter in steel, FIG. 18B shows 2 square laser-drilled holes with eachside being about 50 μm formed in silicon, and FIG. 18C shows severalholes about 50 μm in diameter formed laser-drilled in Kapton.

A technique that permits vent holes 1742 to be formed in the film layer1720 after the film layer 1720 has been bonded to the base 1730eliminates the need to precisely align the film layer with the base,which may thereby facilitate manufacturing. In other words, in a filmlayer that has pre-formed holes, precise alignment of the film layerwith the base during bonding is needed to ensure alignment of thepre-formed holes with the venting chambers. Moreover, a micro-machiningtechnique for forming the holes, such as that described above, forexample, permits the size (e.g., diameter) of the vent holes to bealtered as desired and progressively. This may permit control over thepressure gradient along the fill path during filling of the substrate.

Although the exemplary embodiment of FIG. 17 depicts a single vent hole1742 corresponding to each venting chamber 1790, it should be understoodthat one or more vent holes 1742 may be provided in communication witheach venting chamber 1790. The number of vent holes per venting chambermay be selected based on a variety of factors, including size of thevent holes, desired venting of the gases in the substrate, minimizationof leakage of sample from the substrate, and other factors. Results oftests performed for substrates having differing number of vent holesassociated with each venting chamber are provided below.

Filling tests were performed on substrates defining a 24 sample-chamberarray having a configuration substantially as shown in the partialschematic representation of the substrate 1910 depicted in FIG. 19. InFIG. 19, the arrangement of the sample chambers 1980, inlet channels1975, venting channels 1976, venting chambers 1990, main fluid channel1970, and sample supply inlet 1960 are shown. The substrate used for thetests also included a film layer like the film layer 1720 with ventholes 1742 aligned with the venting chambers 1990. The substrates 1910were made of a COP base covered with an aluminum PSA film layer thatincluded a 5 mm thick aluminum layer with a 1.5 mm thick PSA laminatelayer.

In a first test configuration, a single vent hole of approximately 10 μmin diameter was laser-drilled in the film layer and aligned with eachventing chamber. In a second test configuration, three vent holes ofapproximately 10 μm in diameter were laser-drilled in the film layer andaligned with each venting chamber. In a third test configuration, sixvent holes of approximately 10 μm in diameter were laser-drilled in thefilm layer and aligned with each venting chamber. A syringe pump wasused to supply a red dye fluid to each substrate at a pump speed of 40μl/minute. Red dye was used to assist in observing the flow and fillingin the substrate.

For the first test configuration using a single hole for each ventingchamber 1990, no leakage was observed during filling. For the secondtest configuration using three holes for each venting chamber 1990,single droplet leakage was observed for three vent locations. For thethird test configuration using six holes for each venting chamber 1990,single droplet leakage was observed in two vent locations. Those skilledin the art would understand that the number and/or size of vent holesprovided for each venting chamber may vary based on a variety offactors, including, the sample being introduced, the pressure in thesubstrate, and other factors. Overall, the size and number of vent holesmay be chosen so as to substantially prevent leakage of sample throughthe one or more vent holes and out of the device, while permitting gas(e.g., air) to escape through the one or more vent holes.

Various exemplary embodiments may utilize a hydrophobic, porous filter,substantially in the form of a fiber-like configuration, in lieu of aventing membranes described earlier, to permit gas (e.g, air) to escapethe substrate while preventing sample leakage therethrough. Although theembodiments described below use a hydrophobic, porous fiber member, itmay also be possible to utilize a porous or gas-permeable membranematerial formed into a fiber-like structure. With reference to FIG. 44,an exemplary embodiment of a substrate 4410 for biological sampleanalysis is depicted. The substrate 4410 includes a base 4430 and a filmlayer 4420 covering the base 4430. The substrate 4410 defines a sampledistribution network including an array of sample chambers 4480 in flowcommunication with a plurality of main fluid supply channels 4470 viasample introduction branch channels 4475. Each sample chamber 4480 alsois in flow communication with a main venting channel 4472 via branchventing channels 4476. A hydrophobic, porous fiber 4400 may be placed inthe main venting channel 4472, as shown in FIGS. 44 and 44A. Thus,rather than each sample chamber 4480 terminating in an individualventing chamber, as described in other embodiments herein, a group ofsample chambers 4480 terminates in a common venting channel 4472. Asshown in the close-up view of FIG. 44A, the film layer 4420 may beprovided with vent holes 4425 aligned with the main venting channel4472. Each adjacent pair of chambers 4480 and corresponding ventingchannels 4476 may be associated with a vent hole 4425, as depicted inFIG. 44A. The vent holes 4425 may permit gas to escape through thefibers 4400 and out of the substrate 4410. The vent holes 4425 may beformed via a variety of techniques, including the laser processdescribed above with reference to FIG. 17. In an alternative embodiment,rather than providing vent holes 4425 in the film layer 4420, ventthrough holes may be formed from the venting channel 4472 through thedepth of the base 4430, opening to the bottom of the base 4430 shown inFIGS. 44 and 44A.

The hydrophobic porous fibers 4400 may have a configuration similar tosuch fibers used in the filtration industry to filter impurities fromwater pumped into the fiber at pressures higher than the outside of thefiber. Such fibers permit impurities to flow through the pores of thefiber wall while water is retained. In various other exemplaryembodiments, the fibers 4400 may be in the form of a resilient fibercord that has a porous hydrophobic coating. Similarly, in the case ofuse with a substrate for biological testing, the filter 4400 can permitgas (e.g., air) to pass therethrough while retaining sample. Thus, withthe fibers 4400 in place in the substrate 4110, gas may be permitted topass through the main venting channels 4172 and fibers 4400 and out ofthe substrate 4410 through vent through holes.

FIG. 45 shows an exemplary technique for placing the fibers 4400 in anassembly-line fashion into a plurality of bases 4430 to form substrates4410. A fiber supply roller 4500 may supply a plurality of separatefibers 4400, for example, corresponding to at least the number of mainventing channels 4172 in a substrate. The fibers 4400 may be secured inposition in the main venting channels 4172 in the first base 4430 andthe first base 4430 may move down a belt or other similar device,thereby pulling the fibers 4400 with it. As the bases 4430 move to theright shown in FIG. 45, new bases 4430 to be supplied with fibers 4400are added to the left end. After a desired number of bases 4430 havebeen supplied with fibers 4400, a film layer may be adhered to the bases4430 and the parts cut away from each other in the spaces between theparts shown.

FIGS. 46A-46C show an exemplary embodiment for securing hydrophobicporous fibers in place in venting channels of a base portion of asubstrate. For simplicity, the base 4630 depicted in FIGS. 46A-46C showsonly two rows of sample chambers 4680 in flow communication with acommon main fluid supply channel 4670 and differing main ventingchannels 4672. FIG. 46A depicts the base 4630 prior to the placement ofthe fibers 4600 in the channels 4672. A plurality of weld spots 4650 aredeposited along the length of each of the main venting channels 4672 inpositions between where adjacent venting channels 4676 intersect themain venting channels 4672. By way of example, the weld spots 4650 maybe formed from a low melting point polymer deposited in the channels4672, for example, via an ink-jet type of device. In another example,the base 4630 may be molded with the weld spots 4650. Between the weldspots 4650, vent through holes 4640 may be provided in the base from thechannels 4672 to the bottom of the base 4630 in order to permit gas toescape the substrate 4610. Alternatively, such vent through holes may beprovided in the film layer that covers the base 4630, as has beendescribed herein.

As shown in FIG. 46B, the fibers 4600 may be placed in the channels4672, for example via a fiber supply tool as was described withreference to FIG. 45 above. A heated pressing instrument 4655 may beused to press the fibers 4600 into the channels 4672, preferably whilethe fibers 4600 are held in tension. At the same time, the heatedinstrument 4655 melts the weld spots 4650 to fuse the weld spots 4650and fibers 4600 together at the locations of the weld spots 4650, asshown in FIG. 46C. This melting process may serve to block the pathsbetween the sample chambers 4680 and thus may serve as a sealingmechanism for sealing the chambers 4680. A series of bases 4630 may beformed in this way using the assembly line process discussed in FIG. 45,with the film layers being applied and the substrates being separatedfrom each other by cutting the fibers as described above. In otherembodiments, a heated instrument may be used after the film layer 4620has been applied in order to fuse the weld spots 4650 and fibers 4600together and at the same time bond the film layer 4620 to the base 4630.

In yet further various embodiments, the instead of the weld spots 4650,a two layer laminated material may be used.

FIG. 46D depicts a partial cross-section of the completed substrate 4610with the film layer 4620 adhered to the base 4630. The cross-section inFIG. 46D is taken through a vent through hole 4640. As shown in FIG.46D, the main venting channel 4672 may have a depth that is less than adiameter of the fiber 4600 such that the film layer 4620 presses down onthe top of the fiber 4600 to hold the fiber against the bottom of thechannel 4672 and seal off the vent through hole 4640 to ensure that nosample leaks around the fiber 4600 and escapes through the through hole4640. If needed, the substrate 4610 may be held against a flat plate orthe like during filling to prevent the film layer 4620 from bulgingrather than maintaining a tight seal like that shown in FIG. 46D.

To further improve sealing of the vent through holes 4640 with the fiber4600, a circular seal ring 4642 may be provided around the vent throughhole opening in the channel 4672, as shown in FIGS. 47A and 47B. Thering 4642 may have a raised surface relative to the bottom surface ofthe channel 4672 and provide a flat surface to press against the fiber4600 rather than, for example, a rounded surface of the bottom of thechannel 4672. Further, because the surface of the seal ring 4642 isslightly raised relative to the bottom of the channel 4672 in the areaof the vent through hole 4642, a better seal may be achieved between thefiber 4600 and the vent through hole 4642.

Although FIG. 47B depicts a hollow tubular fiber structure, it should beunderstood that porous hydrophobic fibers in accordance with the presentteachings may have a variety of cross-sectional shapes, including, butnot limited to, for example, a solid circular cross-section (e.g., arod).

FIGS. 48-50 show another exemplary embodiment of a substrate 4810 thatincludes a porous, hydrophobic fiber 4800 for retaining sample in thesubstrate while permitting gas to escape. FIGS. 48A and 48B showopposite sides of the base portion 4830 of the substrate 4810, whileFIG. 48C shows the substrate 4810 including film layer 4820 as viewedfrom the same side as in FIG. 48B. In the embodiment of FIGS. 48A-48C,the base 4830 is transparent and the film layer 4820 may be metallic,such as, for example, an aluminum PSA film layer. However, it should beunderstood that the base 4830 and film layer 4820 may be made of anymaterials described herein as suitable for making a base and film layer.To avoid confusion between features of the substrate 4810 on the nearand far sides, FIGS. 48A and 48B are shown as being opaque.

With reference to FIG. 48A, the substrate 4810 may comprise a base 4830that, together with the film layer 4820, defines a sample distributionnetwork that includes a plurality of sample chambers 4880 which allconnect to a common main fluid supply channel 4870. Each chamber 4880 isin flow communication with the main fluid supply channel 4870 via asample introduction channel 4875. Each chamber 4880 also is in flowcommunication with a venting channel 4876 that leads to a main ventingchannel 4872 provided in the side of the base 4830 facing up in FIG.48B, i.e., opposite to the side in which the other features discussedabove are provided. The substrate 4810 further includes a sample inletport 4860 in flow communication with the main fluid supply channel 4870.An initial portion 4865 of the main fluid supply channel 4870 may have aserpentine configuration so as to permit passive mixing of the sample,for example, of an eluted sample, prior to introducing the sample to theintroduction channels 4875. A more detailed explanation of using aserpentine channel to achieve sample mixing is provided below.

A hydrophobic, porous fiber 4800 may be provided in the main ventingchannel 4872 in a manner similar to that described above with referenceto the exemplary embodiments of FIGS. 44-47. As shown in FIGS. 48C andthe close up views of FIGS. 49 and 50, a film layer 4820 may cover theside of the base 4830 shown in FIG. 48A and a portion of the film layer4820 may wrap around the base portion to cover and seal the channel 4872and fiber 4800. Flow communication between the venting channels 4876 andthe main venting channel 4872 may be provided via a vent through hole4840 that leads from the end of the venting channels 4876 to the ventingchannel 4872. According to various exemplary embodiments, in a mannersimilar to that described above in FIGS. 47A and 47B, the vent throughholes 4840 may terminate in the main venting channel 4872 in a raisedsealing rim 4842 that presses against the fiber 4800, as shown inpartial cross-sectional view of FIG. 49.

Vented air may pass into the porous fiber 4800, which may be in the formof a hollow tube as shown or may have other configurations, as describedabove. The air may pass down the fiber 4800 and/or exit the fiber 4800into the channel 4872 that the fiber 4800 lies in. According to variousembodiments, a single vent hole 4825, shown in FIG. 50, may be providedin the film layer 4820 and aligned with the main venting channel 4872,permitting any air leaving the substrate 4810 to pass therethrough.Providing a single vent hole 4825 may limit the potential of sampleescaping from the substrate due, for example, to improper sealing of thevent passages after filling the substrate 4810. However, it should beunderstood that plural vent holes also may be formed in the film layer4820 to allow air to escape therethrough.

FIGS. 51A and 51B show the opposing sides of another substrate providedwith a porous, hydrophobic fiber venting member according to variousembodiments of the present teachings. The substrate 5110 of FIGS. 51Aand 51B has substantially the same structure as the substrate 4810described above, except that the main fluid supply channel 5170 isprovided in the same side of the base 5130 as the main venting channel5172. The two channels 5170 and 5172 are depicted in the view of FIG.51B and are on a side of the substrate opposite to the chambers 5180,sample introduction channels 5175 and venting channels 5176. As with themain venting channel 5172, flow communication between the introductionchannels 5175 and the main fluid supply channel 5170 may be provided viathrough holes (not shown) in the base 5130. The configuration of FIGS.51A and 51B may permit isolation of the sample chambers 5180 via asealing (staking) mechanism on the detection side (e.g., the side shownin FIG. 51B) of the substrate 5110. This may allow the sealing mechanismto be provided on a portion of instrumentation that is not part of athermocycler and thus may be made of materials that do not need to takethermal properties into consideration.

Providing the sample chambers 4880 and 5180 in two rows, as shown in theexemplary embodiments of FIGS. 48-51 may be advantageous in that all ofthe chambers 4880 and 5180 are positioned at an outer perimeter of thesubstrate 4810 and 5110. This may avoid edge effects that may causeinterior chambers of a substrate to experience differing temperaturesthan temperatures of chambers at a perimeter of the substrate. Thus, allof the chambers may have a substantially uniform temperature, forexample, during thermocycling of the substrate. Even in the case whereone row of chambers is hotter than the other row of chambers if thetemperature difference is uniform between the two rows, the temperatureof the chambers may be uniform. Further, due to the relatively smallsize of the substrates 4810 and 5110, for example, in the configurationshown that includes 16 chambers, a smaller thermal block may be usedwith reduced margin on either side, which may decrease the overall sizeof the instrumentation used for biological testing of the substrates4810 and 5110.

Although the exemplary substrates 4810 and 5110 include an array of tworows of chambers 4880 and 5180, the substrates may be formed with anynumber of chamber rows. By way of example only, the substrates may beformed with four rows of chambers, in which case a main fluid supplychannel may be positioned between a first pair of chamber rows and asecond pair of chamber rows. Two venting channels may then be providedat the two opposite edges of the substrate in conjunction with each ofthe pair of rows of chambers. Any number of rows may be used, with theporous, hydrophobic filters disposed inward from the edges of thesubstrate being secured in position by a separate film or series ofstrips of film on the underside of the substrate. The substrates alsomay include negative template control sections, described in more detailwith reference to the embodiments of FIGS. 26 and 27, which according tovarious embodiments may be provided substantially in the center of thesubstrate array with corresponding sample inlet ports.

The substrates 4810 and 5110 may be assembled in a manner similar tothat described above with reference to FIG. 45, that is, in a continuousfashion by tensioning the fibers 4800 and 5100 from a roll and makingplural substrates 4810 and 5110 in an assembly line. According tovarious embodiments, after applying the film layer to the bases, asdescribed with reference to FIG. 45, the substrates 4410, 4810, and 5110may be left in a continuous strip-like configuration (e.g., withoutseparating the individual substrates) and packaged in a reel 5200, asshown in the exemplary embodiment of FIG. 52. The reel 5200 may have acutter mechanism 5250, similar to a tape reel, in order to separateindividual substrates as desired. According to yet other exemplaryembodiments, the substrates may be left connected to one another andsupplied in an automated manner to a processing instrument, such as, forexample, a thermocycler or the like, in a continuous and/or highthroughput manner. This may permit processing of the substrates withoutan operator handling each substrate individually, which couldpotentially contaminate and/or damage each substrate. Those havingordinary skill in the art would understand how to package any of thesubstrate embodiments herein in a continuous reel mechanism like that ofFIG. 52.

In yet other embodiments, a thermocycler may be configured toaccommodate two sample substrates, for example, substrates 4810 or 5110.A perspective view of such a thermocycler 5300 is depicted in FIG. 53Aand a partial cross-sectional view is depicted in FIG. 53B. Thesubstrates could be processed at both ends of the thermocycler 5300(e.g., the left and right sides) shown in FIG. 53A. As shown in FIG.53B, the thermocycler 5300 may be provided with a heated plate (thermalblock) 5350 having a crowned profile and pressure may be applied to thechamber array by providing pressure on the outer edges of the substrate5310 without transmitting force through the open area (or window) 5305directly over the sample chambers of the substrates. A relatively thin,narrow heated plate 5350 may be used and transmit sufficient clampingforce to the side walls of the substrates rather than through a Peltierdevice or other component.

As discussed above, control over the pressure gradient along the fillpath during filling of the substrate may be provided, for example, bycontrolling the size of vent holes, such as vent holes 1742 provided inthe substrate 1710, as was described in relation to the embodiment ofFIG. 17. Other techniques also may be used, either alone, in combinationwith the vent holes or other substrate configurations in accordance withthe disclosure, and/or in combination with each other, to providecontrol over the pressure gradient during filling of a substrate viapositive pressure. It may be desirable to control the pressure gradientby creating a higher pressure in the venting channels so as to reducethe potential for leakage from the substrate (e.g., through the ventholes in a film layer).

By way of example, the hydrophobicity of the venting channels of thesubstrate may be modified, for example increased, to control thepressure gradient while filling the substrate with sample. Thehydrophobicity may be modified, for example, by adding texture and/orincreasing roughness (e.g., on a nano-scale level) to the surfacedefining the venting channels. Such texturing and/or increasingroughness may be introduced during the injection molding process, forexample, by texturing the mold as desired in the area that forms theventing channels. Other techniques for modifying the hydrophobicity of asurface defining the venting channels may include providing a coating,or chemically treating the surface. By way of example only, Kim et al.,“Nanostructured Surfaces For Dramatic Reduction Of Flow Resistance InDroplet-Based Microfluidics,” IEEE 2002, hereby incorporated byreference in its entirety herein, teaches one technique for providingnanostructures on a surface to alter hydrophobicity. It is envisionedthat the hydrophobicity of all or a portion of the venting channels maybe altered.

According to various embodiments, the venting channel configuration(e.g., geometry) also may be modified in order to control the pressuregradient during filling of the substrate. For example, the ventingchannels may be provided with a region of reduced cross-section so as toincrease the pressure within the venting channel and reduce thepotential for leakage. With reference to the exemplary embodiments ofFIGS. 20A and 20B, a sample chamber 2080 and corresponding ventingchannel 2000 in flow communication with the chamber 2080 is depicted. Asshown, the venting channel 2000 may be provided with a reducedcross-section R, for example, toward an end of the venting channel 2000that leads to the venting chamber 2090. In the exemplary embodiment ofFIG. 20A, the reduced cross-section R is achieved by narrowing the sidewalls defining the channel 2000. In the exemplary embodiment of FIG.20B, the reduced cross-section R is achieved by raising the bottomsurface of the channel 2000 at the location R in comparison to theremainder of the bottom surface of the channel. In other words, thedepth of the channel 2000 is less at the location than the depth of theremainder of the channel.

According to various embodiments, for example, when filling amulti-chambered substrate via positive pressure (e.g., via pumping,syringe, etc.), it is desirable to know when to stop the filling oncethe various chambers and channels have been filled in order to controlover-pressurization and/or sample leakage. An exemplary mechanism fordetermining when to stop filling the substrate includes providingoptical sensors in association with the sample chambers. The sensors,which in an exemplary embodiment may be an optical sensor including aphotodiode and LED, can detect the presence of the sample by adifference in the index of refraction and send a signal to stop thefilling process (which may occur either manually or automatically). Dueto potential increased costs and manufacturing complexity associatedwith such a sensor/feedback mechanism, it may be desirable to provide arelatively simple substrate design configured to passively andautomatically stop sample delivery so as to avoid over-pressurizationand/or leakage of the substrate.

FIGS. 21A-21C schematically depict exemplary steps of filling amulti-chambered substrate that includes a venting mechanism, such as,for example, either a vent membrane or vent hole, as have been describedabove. It should be noted that FIGS. 21A-21C are simplified for thepurposes of showing the principles of filling channels and/or chambersof a substrate and leakage of fluid that may occur due toover-pressurization. Thus, in the figures, only a single channel isillustrated with a vent positioned at a distal end of the channel.

Referring to FIG. 21A, a volume of sample S is delivered, for example,via positive pressure, at an end of the channel 2190 (or chamber)opposite to an end at which vent membrane 2150 is disposed. The dashedarrows in FIGS. 21A-21C indicate the direction of sample delivery andmovement through the channel 2190, with the shaded area representing thesample S and the nonshaded area representing gas (e.g., air). Thechannel 2190, in an exemplary configuration, may be defined by a baseportion 2130 and a film layer 2120, with the film layer 2120 comprisinga vent hole (not shown) positioned beneath the membrane 2150 to permitgas to escape therethrough. In FIG. 21A, as the sample S is forced viapressure through the channel 2190, gas (e.g., air) residing in thechannel 2190 is compressed and passed out of the channel 2190 throughthe vent hole (not shown) and the gas-permeable membrane 2150, as shownby the solid arrow proximate the membrane 2150. As the sample Scontinues to move downstream in the channel 2190 (e.g. toward themembrane 2150), gas that is between the sample S and the end of thechannel 2190 continues to be released through the vent membrane 2150, asshown in FIG. 21B.

Eventually, due to the continued positive pressure applied at the end ofthe channel 2190 proximate the dashed arrow, as shown in FIG. 21C, thesample S reaches a location in the channel 2190 corresponding to thevent hole (not shown) and membrane 2150 (e.g., the end of the channel2190 opposite the end to which pressure is applied). If further pressureis applied after the sample S reaches the position shown in FIG. 21C,the channel 2190 becomes over-pressurized and the sample S may leak outof the vent hole and membrane 2150. For example, in the case of agas-permeable, liquid impermeable membrane 2150, the membrane may burstdue to over-pressurization and/or sample S may leak around the edges ofthe membrane 2150, as depicted by the arrow in FIG. 21C.

In FIGS. 21A-21C, it should be understood that the membrane 2150 may beeliminated and a vent hole of micro-size may be used instead, asdescribed, for example, with reference to the embodiment of FIGS. 17 and19.

Leakage of sample out of the substrate may depend upon various factors,including, for example, the configuration of the substrate, the appliedpressure, the method of pressure generation (e.g., via syringe, pump,constant pressure source, etc.), properties of the membrane materialand/or configuration of vent holes, and other factors that may influencethe extent to which the substrate becomes over-pressurized duringfilling with sample.

According to various embodiments, providing an additional vent upstreamof the vent 2150 of FIGS. 21A-21C may alleviate over-pressurization andleakage. FIGS. 22A-22C schematically depict the filling process thatoccurs for the channel 2190 of FIGS. 21A-21C when an additional, bypassventing mechanism 2250 is placed upstream of the vent membrane 2150.

In FIG. 22A, like in FIG. 21A, sample S is delivered, for example, viapositive pressure, at an end of the channel 2190 (or chamber) oppositeto an end at which gas-permeable vent membrane 2150 is disposed. Thedashed arrows in FIGS. 21A-21C indicate the direction of sample deliveryand movement through the channel 2190, with the shaded area representingthe sample S and the nonshaded area representing gas (e.g., air). Thechannel 2190, in an exemplary configuration, may be defined by a baseportion 2130 and a film layer 2120, with the film layer comprising avent hole (not shown) positioned beneath the membranes 2150 and 2250 topermit gas to escape therethrough. Alternatively, as described withreference to FIGS. 21A-21C above, the membranes 2150 and 2250 may beeliminated, and a micro-sized vent hole in the film layer used instead.

In FIG. 22A, as the sample S is forced via pressure through the channel2190, gas (e.g., air) residing in the channel 2190 is compressed andpassed out of the channel 2190 through the vent holes (not shown) andthe gas-permeable membranes 2150 and 2250, as shown by the solid arrowsproximate the membranes 2150 and 2250. As the sample S continues to movedownstream in the channel 2190 (e.g. toward the membrane 2150), gas thatis between the sample S and the end of the channel 2190 continues to bereleased through the vent membrane 2150, with no gas being releasedthrough the membrane 2250 while the sample S moves into the region ofthe channel 2190 aligned with the membrane 2250, as shown in FIG. 22B.

Eventually, due to the continued positive pressure applied at the end ofthe channel 2190 proximate the dashed arrow, as shown in FIG. 21C, thesample S reaches a location in the channel 2190 corresponding to thevent hole (not shown) and membrane 2150 (e.g., the end of the channel2190 opposite the end to which pressure is applied). In the additionalupstream venting arrangement depicted in FIG. 22, however, if furtherpressure is applied after the sample S reaches the position shown inFIG. 22C, gas trapped in the channel 2190 upstream of the sample S maybe released through the membrane 2250 and corresponding vent hole (notshown), as indicated by the solid arrow proximate the membrane 2250. Byreleasing the gas through the additional upstream venting mechanism(e.g., membrane 2250 in FIGS. 22A-22C), over-pressurization of thechannel 2190 may be prevented and the sample movement in the channel2190 will stop and the sample S will remain in the position shown inFIG. 22C without leaking from the channel 2190.

In order to provide the proper functioning of the upstream bypassventing mechanism, as described with reference to FIGS. 22A-22C,however, the location of the upstream venting mechanism and the volumeof the sample supplied must be selected so as not to causeover-pressurization or incomplete sample delivery. Examples of howover-pressurization and incomplete delivery may occur if the samplevolume and upstream venting position are not chosen appropriately areschematically depicted in FIGS. 23A and 23B, respectively.

Referring to FIG. 23A, incomplete sample delivery may occur if thelocation of the upstream venting mechanism 2355 and the volume ofdelivered sample S are not selected appropriately. In other words, thesample S will not reach and fill the end portion of the channel (orchamber) 2390 and gas (e.g., air) will become trapped underneath theventing mechanism 2350 downstream of the sample S. The situation in FIG.23A may occur when the amount of sample S supplied to the channel 2390and the location of the upstream bypass vent mechanism 2355 are suchthat the sample S advances past the vent mechanism 2355 prior toreaching the vent 2350 at the end of the channel 2390 where it isdesired to collect the sample S. In this situation, as depicted in FIG.23A, as positive pressure is supplied to the channel 2390, shown by thedashed arrow, the sample S is moved within the channel 2390 toward thevent mechanism 2350. However, as the sample S moves past the upstreamvent mechanism 2355, the sample front has not yet reached the ventmechanism 2350, but continued application of pressure causes gas infront of the sample S (i.e., to the left of the sample S in FIG. 23A) toescape through the upstream vent mechanism 2355. This upstream ventingof gas results in the pressure becoming equalized with the atmospheredespite the continued application of pressure in the channel 2390. Dueto the pressure equalization, there is no pressure to cause furtheradvancement of the sample S within the channel 2390, thus resulting inincomplete delivery of the sample S to the desired location (e.g., theend of the channel beneath the vent mechanism 2350 in FIG. 23A).

On the other hand, as depicted in FIG. 23B, the location of the upstreamventing mechanism 2355 and the amount of sample S delivered to thechannel 2390 may be selected such that the sample S completely blocksboth venting mechanisms 2355 and 2350 once the sample S has advancedthrough and reached the end of the channel 2390. In this situation,continued application of pressure to the channel 2390, as indicated bythe dashed arrow in FIG. 23B, may result in sample S leaking from theventing mechanism 2350, as shown by the solid arrow in FIG. 23B. Sampleleakage through the venting mechanism 2350 and/or 2355 may occursubstantially as described with reference to FIG. 21C.

Referring now to FIG. 24, an exemplary embodiment of a multi-chambersubstrate 2410 may include an upstream venting mechanism that protectsagainst over-pressurization and leakage, as described in FIGS. 22A-22C,while also including features that avoid the problems described in FIGS.23A and 23B. As shown in FIG. 24, the substrate 2410, which may includea base and film layer in accordance with various embodiments of thedisclosure, defines a plurality of sample chambers 2480 forming anarray. The chambers 2480 are in flow communication with a plurality ofsample introduction chambers 2475, which distribute sample supplied tothe substrate 2410 via a main fluid channel 2470. The chambers 2480 alsoare in flow communication with venting chambers 2490 via ventingchannels 2476. The venting chambers 2490 are associated with ventingmechanisms (not shown), such as, for example, the various membraneembodiments or micro-sized vent holes described herein.

Upstream of the chambers 2480, the substrate 2410 is provided with aventing mechanism 2455, which may be, for example, in the form of a venthole in a film layer of the substrate 2410 covered with a ventingmembrane. This upstream venting mechanism 2455 may allow gas (e.g., air)to escape from the substrate 2410 after the various channels andchambers have been filled such that over-pressurization and sampleleakage out of the venting mechanisms associated with the ventingchambers do not occur.

In accordance with the exemplary embodiment of FIG. 24, the substrate2410 also defines an overfill channel 2475 and overfill chamber 2495.The overfill channel 2475 leads from the downstream end of the mainfluid channel 2470 and terminates in the overfill chamber 2495. Thepurpose of the overfill channel 2475 and overfill chamber 2495 is toprovide a collection reservoir for the sample so as to ensure thatcomplete delivery of the sample to the chambers 2480 and ventingchambers 2490 occurs. Providing an overfill chamber 2495 of sufficientsize allows for a sufficient volume of sample S to be loaded into thesubstrate 2410 to ensure complete delivery of the sample S, without arisk of overfilling and/or overpressurizing the substrate 2410 such thatleakage may occur.

The overfill channel 2475 may have a smaller cross-sectional area thanthe main fluid channel 2470. The smaller cross-section will increase thefluidic resistance (e.g., pressure) encountered by the sample S as itfills the substrate 2410. As such, the overfill chamber 2495 will fillonly after the remaining chambers 2480 and 2490 and channels 2470, 2420,and 2400. In various embodiments, rather than a straight overfillchannel of reduced cross-section, an overfill channel having aserpentine configuration may be used as depicted in FIG. 24 and/or acombination of serpentine configuration and reduced cross-section may beused. The serpentine configuration can lengthen the overfill channel incomparison to a straight overfill channel substantially withoutincreasing the overall size of the substrate. The lengthening andserpentine configuration of the overfill channel also may function toincrease fluidic resistance encountered by the sample such that theoverfill chamber fills after the remaining portions of the substrate.

According to various embodiments, the inlet sample volume requirementmay be calculated as follows to ensure that all of the sample chambers2480 are filled and the substrate is not overpressurized. Assuming thatthe substrate 2480 has 24 sample chambers, as shown in FIG. 24, theinlet sample volume=(the volume of all 24 chambers 2480)+(the volume ofall 24 venting chambers 2490)+(the volume of the main fluid channel2470)+(the volume of the sample introduction channels 2420)+((the volumeof the overfill chamber 2495)/2) Thus, the sample volume tolerance usingthe above inlet sample volume is ½ the volume of the overfill chamber,which will allow the volume to fill all of the sample chambers, withoutover-pressurizing the device.

According to various embodiments, aspiration of sample into the chamberscan be assisted by moving the overfill chamber to the inlet to provideprotection against over-aspirating the sample.

As discussed above, in various embodiments, it may be desirable tointegrate sample preparation with a multi-chamber array substrate. Forexample, it may be desirable to elute nucleic acid from a membrane andsupply the eluted sample volume directly to a substrate. Before fillingthe sample chambers with the eluted sample, however, it is desirable toensure that the eluted sample has been sufficiently mixed tosubstantially homogenize the concentration of the sample prior tofilling the sample chambers. If the eluted sample is not sufficientlyhomogenized, a concentration gradient may result in the substratechambers. For example, the concentration of nucleic acid may be higherin upstream chambers than in downstream chambers of the substrate. Sucha concentration gradient in the substrate may impair detection,quantization (e.g., for gene expression) and/or analysis of thebiological sample being tested.

To mix the eluted sample so as to obtain a substantially homogenizedconcentration, an external mixing force, for example, via a vortex orthe like, may be applied to the collected sample and the mixed samplemay then be introduced into the substrate. In an alternative embodiment,however, it may be desirable to provide a mechanism for mixing elutedsample as part of the substrate itself. FIG. 25 shows an exemplaryembodiment of a substrate configuration that provides for mixing ofeluted sample within the substrate itself prior to the eluted samplebeing loaded into the sample chambers. In the exemplary embodiment ofFIG. 25, passive mixing of the sample may occur in the substrate via arelatively simple design and without moving parts.

In the multi-chamber array substrate 2510 shown in FIG. 25, a tube 2505configured to collect eluted sample (e.g., a sample of nucleic acideluted from a membrane) is positioned in flow communication with asample inlet port 2560 of the substrate 2510. The tube 2505 may collecteluted sample prior to any mixing, for example, nucleic acid sampleeluted directly from a membrane. The substrate 2510 includes an array ofsample chambers 2580, venting chambers 2590, sample introductionchannels 2575, venting channels 2576, and a main fluid channel 2570,similar to other substrate embodiments described herein.

In addition to the various features listed above, the substrate 2510also defines a serpentine mixing channel 2565 that connects the inletport 2560 and the main fluid channel 2570 in flow communication witheach other. The serpentine channel 2565 serves to lengthen the distancethe sample travels between being supplied to the inlet port 2560 andfilling the sample chambers 2580. By increasing the distance, and thustime, the sample travels prior to filling the chambers 2580, diffusionmay be increased in the sample thereby mixing the sample and promotinghomogenization of the sample concentration. In other words, moving aplug of liquid, such as a volume of eluted sample, through a channel ofsufficient length prior to introducing the sample into the samplechambers may take advantage of the recirculation patterns that occuralong the axis of the microfluidic channel that results from the plug ofliquid having a parabolic velocity profile with substantially flatmenisci at both ends of the plug. With a long enough mixing channel, andthus time, such recirculation patterns may act to mix the sample plugand provide a substantially uniform concentration prior to the samplebeing introduced into the sample chambers. Enhanced mixing may alsooccur by providing the mixing channel 2565 with sharp corners ratherthan rounded corners and/or by applying various surface finishesconfigured to enhance mixing.

Thus, the mixing channel 2565 provides a passive mixing feature that isintegral with the substrate 2510. This permits direct loading of thesubstrate 2510 with eluted sample without prior mixing, while ensuringthat the sample filling the chambers 2580 will have a substantiallyuniform concentration for all of the chambers 2580. The serpentinemixing channel 2565 may be relatively large in comparison to the mainfluid channel 2570. By way of example only, the main fluid channel 2570may be approximately 150 μm wide by approximately 50 μm deep, while thewidth of the mixing channel 2565 may range from approximately 1 mm toapproximately 2 mm and have the same depth as the main fluid channel2570.

A straight mixing channel may be used rather than the serpentine mixingchannel shown in FIG. 25, however, using a straight channel havingapproximately the same length of the serpentine channel may increase theoverall dimensions of the substrate 2510. Thus, the serpentineconfiguration provides a benefit of providing a sufficient length overwhich diffusion of the eluted sample can occur, without substantiallyincreasing the overall dimensions of the substrate. Further, the bendsin the serpentine channel configuration may promote additional mixing ofthe sample as it travels through the channel.

Yet another exemplary embodiment of a multi-chamber array substrate 2610is depicted in FIG. 26. The view of the substrate 2610 shows the variousfeatures of the base 2630 of the substrate that, together with a filmlayer, as has been described for various substrate embodiments above,form a sample fluid distribution network including main fluid channels2670 a, 2670 b, and 2671, inlet channels 2672 leading from the mainfluid channels to sample chambers 2680, and venting channels 2600leading from sample chambers 2680 to venting chambers 2690. Thus, theview in FIG. 26 is through a film layer applied over the side of thebase 2630 that has openings defining the various features describedabove. Further features of the substrate 2610 include sample inlet ports2660 for supplying sample to the substrate 2610 and to main fluidchannels 2670 a, 2670 b, and 2671, overfill chambers 2695 and overfillchannels 2675 leading from main fluid channels 2670 b and 2671 to theoverfill channels 2695. According to various exemplary embodiments, thesubstrate 2610 also may include indexing holes 2621 to help align andposition the substrate 2160 in various biological testing apparatuses,such as, in conjunction with thermal blocks and the like for performingPCR or other biological analysis.

The substrate 2610 of FIG. 26 may be made of a variety of materials forthe base and film layers, including any of the materials that have beendiscussed above. In particular, the materials chosen may be PCRcompatible materials. By way of example, the base 2630 may be mademolded from COP (e.g., ZEONOR 1420R) and the film layer that togetherwith the base 2630 defines the sample distribution network (e.g., thesample chambers, channels, venting chambers, etc.) may be an aluminumPSA layer. In addition, it is envisioned that various sealing, venting,and mixing mechanisms that have been described above may be used incombination with the substrate depicted in FIG. 26 and those havingskill in the art would understand based on the teachings herein how tocombine those mechanisms and/or various structural configurationsassociated with those mechanisms with the embodiment of FIG. 26. By wayof example only, in various embodiments, the substrate of FIG. 26 may becombined with any of the venting mechanisms illustrated in FIGS. 14-16.That is, the base 2630 of the embodiment of FIG. 26 may replace the base1430 shown in FIGS. 14-16 and be combined with the other componentsshown in those figures, including the film layer 1420, the film layer1425, and any of the membranes 1450, 1550, and 1650. Other structuralaspects of the substrate 2610 shown in FIG. 26 are discussed below inmore detail.

As noted above, the exemplary embodiment of FIG. 26 includes two inletfluid supply ports 2660. One of the ports 2660 is in flow communicationwith main fluid supply channels 2670 a and 2670 b and the other inletport 2660 is in flow communication with main fluid supply channel 2671.Main fluid supply channels 2670 a and 2670 b are in parallel connectionwith each other and the inlet port 2660, however, the inlet port 2660also may supply fluid to main fluid channels that are seriallyconnected, for example, as shown in the exemplary embodiment of FIG. 27.The main fluid supply channels 2670 a and 2670 b are configured tosupply fluid (e.g., biological sample) to a first group of samplechambers 2680 (e.g., 16 chambers in FIG. 26) and the main fluid supplychannel 2671 is configured to supply sample to a second group ofchambers 2680 (e.g., 8 chambers in FIG. 26). The main fluid supplychannels 2670 a and 2670 b and the first group of chambers 2680 are notin flow communication with the main fluid supply channel 2671 and thesecond group of chambers 2680.

This configuration of two inlet ports 2660 for supplying two differingsample chamber networks that are not in flow communication with eachother permits two differing samples to be supplied to the substrate 2610and/or differing biological testing (analysis) to be performed withinthe same substrate 2610. Moreover, according to various embodiments, thedual fluid distribution network provided in the substrate 2610 mayprovide a negative template control mechanism in order, for example, totest for false positives. By way of example, the inlet portion 2660connected to the main fluid channels 2670 a and 2670 b may be suppliedwith a biological sample for which PCR analysis may be desired, whilethe inlet port 2660 in flow communication with the main fluid supplychannel 2671 may be supplied with a blank sample (such as, for example,an elution buffer such as deionized water or Tris HCl). Analysis, suchas via optical detection (for example, by detection of a fluorescentsignal), of both groups of sample chambers may be performed and if asignal is detected in the sample chambers filled with the blank sample,this may indicate that the substrate is contaminated or otherwisesusceptible to giving a false positive result.

Although the exemplary embodiment of FIG. 26 depicts 16 sample chambersin flow communication with one inlet port 2660 and 8 sample chambers inflow communication with the other inlet port 2660, any number of samplechambers may be provided in flow communication with each inlet port.However, when using one group of sample chambers and corresponding inletport as a negative template control, it may be desirable to provide lesssample chambers than for a group of chambers and corresponding inletport being used for analysis of biological sample. It also should beunderstood that each inlet port may supply more than one main fluidchannel, which may be connected either in parallel or serially. Inaddition, more than two sample inlet ports may be provided and thus morethan two groups of sample chambers may be supplied with differingsamples.

As mentioned above, for an inlet supply port supplying sample to morethan one main fluid channel, those main fluid channels may be connectedeither serially or in parallel. The exemplary embodiment of FIG. 27depicts a substrate 2710 similar to that of the substrate 2610 of FIG.26, with the exception that the main fluid channels 2770 a and 2770 bthat supply the group of 16 sample chambers 2780 are connected in seriesrather than in parallel. Like the substrate 2610, the substrate 2710includes two inlet supply ports 2760 that are configured to supplydiffering fluids to two differing groups of sample chambers 2780, andthus can provide a negative template control as discussed above. Basedon studies performed, providing the configuration of FIG. 26, whereinthe main fluid channels 2670 a and 2670 b are connected in parallelrather than serially like the main fluid channels 2770 a and 2770 b,permits faster filling of the substrate. For example, for machinedsubstrate prototypes having configurations similar to the embodimentsdepicted in FIGS. 26 and 27, using an applied pressure of 2 psi toperform the filling, the embodiment of FIG. 26 filled in 15 secondswhile the embodiment of FIG. 27 filled in 4 minutes. At an appliedpressure of 5 psi, the embodiment of FIG. 26 filled in 6 seconds and theembodiment of FIG. 27 filled in 22 seconds.

The exemplary embodiment of FIGS. 26 and 27 also include two overfillchambers 2695 and 2795 and two overfill channels 2675 and 2775associated with each group of sample chambers 2680 and 2780. Eachoverfill channel 2675 leads respectively from the main fluid supplychannels 2670 b and 2671 to the overfill chambers 2695. Each overfillchannel 2775 leads respectively from the main fluid supply channels 2770b and 2771 to the overfill chambers 2695. In a manner similar to thatdescribed above with reference to FIG. 24, the overfill chambers 2695and 2795 and overfill channels 2675 and 2775 act to protect againstoverfilling, and thus over-pressurization of the substrate whileensuring sufficient filling of all of the sample chambers 2680 and 2780with fluid.

An upstream venting mechanism in conjunction with the inlet supply ports2660 and 2760 may be provided in order to protect against overfill andover-pressurization of the substrate, as discussed above with referenceto FIGS. 22 and 24, for example. The upstream venting mechanism may bein the form of any of the venting mechanisms in accordance with theteachings herein, including, but not limited to, the venting mechanismof the embodiment of FIGS. 2A and 2B, the venting mechanism of theembodiment of FIG. 13, the backside venting mechanisms discussed withreference to the embodiments of FIGS. 15-16, and the venting mechanismof the embodiment of FIG. 17. In an exemplary aspect, the ventingmechanism may be a vent hole provided in the film layer that covers theopenings of the various fluid distribution features of the base (e.g.,the film layer that together with the base forms the fluid distributionnetwork of chambers and channels) and a gas permeable or porous membrane(e.g., hydrophobic membrane) situated over the inlet ports 2660 and2760. Further, rather than positioning the upstream venting mechanismover the inlet ports 2660 and 2760, the upstream venting mechanism couldbe provided in conjunction with the fluid channels leading from theinlet ports 2660 and 2760 at a location proximate the inlet ports 2660and 2760.

Using an upstream venting mechanism, the sample volume that may be usedto fill the sample chambers 2680 and 2780 associated with the main fluidsupply channels 2670 a, 2670 b and 2770 a, 2770 b may range from aminimum determined by adding the total volume of the sample chambers(the 16 chambers in the case of FIGS. 26 and 27), the total volume ofthe venting chambers, the main fluid supply channels, the inletchannels, and the venting channels associated with those chambers, andthe volume of vent through holes (if any, for example, if the substrateof FIGS. 26 and 27 has a configuration like those shown in one of FIGS.14-16) associated with the venting chambers and inlet supply portfeeding the first group of chambers. The sample volume maximum may becalculated by adding the above volumes to the volume of the overfillchamber. According to an exemplary embodiment, assuming that for thefluid distribution networks associated with the group of 16 chambers ofFIGS. 26 and 27 that the volume of each sample chamber 2680 and 2780 is1.35 μL the volume of the overfill chamber 2695 and 2795 is 5.09 μL, thetotal volume of the main fluid channels 2670 a, 2670 b and 2770 a, 2770b, the inlet channels 2670 and 2770 and venting channels 2600 and 2700associated with those main fluid channels, and the venting chambers 2690and 2790 associated with the 16 sample chambers is 1.40 μL, the samplesupply volume may range from 24.53 μL to 29.62 μL.

According to other exemplary embodiments, instead of or in addition toproviding an upstream venting mechanism to protect againstover-pressurization of the substrate, optical detection of samplereaching the overfill chambers 2695 and 2795 may be implemented. In anexemplary aspect, a dried, colored, fluorescence dye (e.g., a red dye)may be deposited in the overfill chambers 2695 and 2705, for example,proximate an inlet of the chambers 2695 and 2795. Thus, when the samplebegins filling the overfill chambers 2695 and 2795, an optical detectionmechanism may detect a change in color in the overfill chamber 2695 and2795 and a feedback control mechanism may send a signal indicating tostop the application of pressure used for filling the substrate 2610 and2710. For example, a feedback signal may be sent to a pressure-providingdevice (e.g., a pump, syringe, etc.) to automatically stop the pressurebeing used to fill the substrate and/or to an individual to manuallystop the pressure.

The optical detection system may, for example, include an optical coverover the substrates 2610 and 2710 such that only the sample chambers2680 and 2780, and the outlet portion of the overfill chambers 2695 and2795 are viewable by an optical reading mechanism. The optical detectionsystem may use, for example, an LED beam to illuminate the overfillchambers 2695 and 2795 and the optical reading mechanism may monitor theoverfill chambers 2695 and 2795 near their respective outlets 2696 and2697 for a change in fluorescence. After all of the chambers 2680 and2780 have been filled, the sample will move into the overfill chambers2695 and 2795, dissolve the predeposited dye and carry it to the outlets2696 and 2796. The optical reading mechanism may then detect afluorescence signal change and send a feedback signal, for example, toan operator or a filling device, to stop the application of pressure forsupplying sample to the substrate 2610 and 2710. In other embodiments,the detector could detect the presence of an internal standard in themastermix mixed with sample (which may be ROX), rather than spottingadditional dye in the overfill chambers.

It has been observed that flowing deionized water into an empty chamberalso causes a signal increase, which may be contributed by air and waterhaving differing optical background signals and/or by the meniscus ofthe traveling water causing a signal change through both reflection anddiffraction effects. Thus, in various exemplary embodiments, rather thanusing a red dye, LED beam, and fluorescence detecting mechanism in theoverfill chamber, an optical sensor configured to detect the presence ofliquid may be used. By way of example, a refractive index sensor may beused to detect liquid filling the overfill chamber. Because therefractive index of water (e.g., sample) differs from that of air, thelight is deflected in a way that differs when the sample enters anoverfill chamber and can be recognized by the detector. When thedetector senses the change, a signal can be sent to stop the applicationof pressure and supply of sample.

Yet further exemplary embodiments for detection of the presence ofsample in an overfill chamber include the use of a capacitance sensor orthe use of an infrared sensor. Regarding the former, a capacitancesensor may be used to measure the capacitance between the film layer(e.g., an aluminum film layer) covering an overfill chamber and theopposite side of the substrate at the location of the overfill chamber.Since the dielectric constant of water is much greater than air, whensample fills the overfill chamber, the sensed capacitance may change andthe capacitance sensor may send a signal indicating to stop filling(e.g., pressure application to) the substrate. FIG. 37 depicts anexemplary embodiment of using a capacitance sensor to detect thepresence of sample in an overfill chamber 3895 in a substrate 3810. InFIG. 37, an electrode 3801 may be positioned on a side of the substrate3810 opposite to the side of a film layer 3820, for example, an aluminumfilm layer. The electrode 3801 may be disposed on the substrate 3810 ormay be part of an instrument cover or the like that clamps the substrate3810 during filling. The aluminum film layer 3820 may be connected to avoltage supply to serve as a second electrode. In the case where thefilm layer is not a metal, another electrode could be positionedunderneath the film layer 3820 similar electrode 3801. A voltage may beapplied between the two electrodes (e.g., 3801 and the aluminum filmlayer 3820 in FIG. 37) and a capacitance of the chamber 3895 may besensed. In various embodiments, the voltage may be applied as an ACfield, and the capacitance may be detected as a phase shift, as well aspermitting multiple readings, instead of the single change that would beregistered with a DC field. An additional, optional electrode 3802 maybe positioned adjacent, e.g., downstream, of the electrode 3801 and overthe overfill chamber 3895 to use as a reference and a differentialmeasurement may be made to increase the accuracy of the capacitancemeasurement. According to various embodiments, a conductivity detectorcan be used where the sample liquid is permitted to come into directcontact with the electrodes.

In various other embodiments, an infrared sensor may be used to detectsample filling of an overfill chamber. Because water and air havediffering absorbance in the infrared range, infrared absorbance and/orreflection may be measured at the overfill chamber to detect thepresence of sample (which contains water) entering the chamber. Forexample, in the case of an aluminum film layer covering the base of asubstrate, infrared reflection off the aluminum layer over an overfillchamber may be detected.

Those having skill in the art will recognize various other detectionmechanisms that may be used to detect the presence of the sample fillingthe overfill chamber, and the exemplary embodiments above should not beconstrued as limiting. Also, it should be understood that the variousoptical detection mechanisms described with reference to the embodimentsof FIGS. 26 and 27 may also be applied to the exemplary embodiment ofFIG. 24.

Using an optical detection mechanism, the volume of sample that may besupplied to the sample distribution networks associated with the firstgroup of sample chambers 2680 and 2780 (the 16 chamber group) of thesubstrates 2610 and 2710 may range from a minimum determined by addingthe total volume of the sample chambers (the 16 chambers in the case ofFIGS. 26 and 27), the total volume of the venting chambers, the mainfluid supply channels, the inlet channels, and the venting channelsassociated with those chambers, the total volume of the vent throughholes (if any) associated with the venting chambers and inlet supplyport feeding the first group of chambers, and half of the volume of theoverfill chamber associated with the first group of chambers. The samplevolume maximum may be determined in the same manner as the sample volumemaximum when using the upstream venting mechanism approach, discussedabove. Thus, assuming the various volume values as discussed above, thesample volume supplied to the first group of chambers 2680 and 2780using optical detection as an over-pressurization/overfill protectionmechanism may range from 27.08 μl to 29.62 μl.

Whether implementing the upstream venting mechanism approach or theoptical detection approach, using an overfill chamber and channel, asdescribed in the embodiments of FIGS. 24, 26, and 27 helps to ensurethat sufficient filling of the sample chambers occurs withoutover-pressurization, and potential leakage, of the substrate. Further,this protection against underfilling and over-pressurization can occurwith a relatively large tolerance of the input sample volume. In otherwords, a precise amount of sample does not need to be determined andused to fill the substrate such that all of the sample chambers arefilled, but over-pressurization does not occur. Rather, there is avolume range that may be used while still protecting againstunderfilling of the chambers and over-pressurization of the substrate.

Although various exemplary substrate embodiments described above andshown in the figures depicted partial views, schematic views, orsubstrates defining a 12-chamber, 16-chamber, or 24-chamber array, itshould be understood that the various configurations and features ofthose embodiments can be applied to substrates of varying sizes andchamber arrays, including, for example, substrates definingmulti-chamber arrays including various number of chambers, including,but not limited to, 12, 24, 36, 48, 96, 192, 384, 3072, 6144, or moresample chambers. In an exemplary configuration, the various substratesdescribed above can have dimensions of, for example, about 127.0millimeters by about 85.7 millimeters and define 384 sample chambers.

In some cases, for example when performing biological testing using asubstrate defining a chamber array that differs from a conventionalsubstrate, it may be desirable to use existing instrumentation for suchconventional substrate configurations to perform biological analysis onsubstrates of other sizes. For example, at least some substrates inaccordance with the present teachings may define 12-, 16-, or 24-chamberarrays, which have fewer chambers, and thus require fewer assays, thanare available in a conventional low density array substrate (e.g., asubstrate including a 96-chamber or 384-chamber array). When performingbiological testing, such as, for example, real-time PCR, on thesubstrates with fewer chambers, a solution may be to perform testing onmore than one substrate and thereby enable analysis of multiple samplesduring the same testing step. However, this may result in a mismatchbetween the number of samples presenting for analysis and the number ofpositions available on a substrate. Further, using a low density arraysubstrate that does not use all of the chambers available may not bedesirable since once subject to biological analysis, the substrates maynot be suitable for further use. Also, in some cases, it may bedesirable to subject one or more samples to differing assay panels(e.g., biological analysis) during a single processing routine (e.g., asingle real time PCR assay step).

In order to achieve at least some of the desirable features above and inaccordance with various embodiments of the present teachings, substratesthat define a subset of chambers (e.g., “subcards”) of a standard lowdensity array substrate (e.g., 96- or 384-chamber array) may be providedthat are configured to be combined and positioned into a holding fixturecompatible for use with standard low density array testinginstrumentation. FIGS. 54-56 show schematic perspective views of variousholding fixtures that may be used to hold one or more subcards inaccordance with exemplary embodiments.

The holding fixtures 5400 and 5600 of FIGS. 54-56 are in the form of a4-sided frame with an array of openings 5401 and 5601 that correspond tolocations of sample chambers on subcards. One or opposing sides of theholding fixtures 5400 and 5600 may define a slot configured to receiveone or more subcards to be inserted into the fixture when performingbiological testing, for example, real-time PCR. In the holding fixtureof FIG. 54, 4 16-chamber array substrates (subcards) 5410 are showninserted into the holding fixture 5400. The holding fixture 5400 isconfigured to hold a number of substrates that combined total a96-chamber array. That is, the holding fixture 5400 includes an array of96 openings 5401. FIG. 55 shows an exemplary embodiment of the use ofthe holding fixture 5400 to hold 4 16-chamber array subcards 5510 thatinclude sample processing modules 5550 connected to the subcards 5510.In various exemplary embodiments, the sample processing modules 5550 maybe detachable from the subcards 5510 prior to performing biologicaltesting.

FIG. 56 depicts an exemplary embodiment of a holding fixture 5600configured to hold subcards 5610 including 24-chamber arrays, with eachsubcard 5610 including 4 chambers across the substrate and 6 chambersdown the substrate. The holding fixture 5600 may define slots atopposing ends to receive 4 subcards having a configuration like subcards5610. The subcards 5610 shown in FIG. 56 include sample processingmodules 5650, which may be detachable. It should be understood that thesubcards without such sample processing modules also may be used.

The configurations of the subcards and holding fixtures of FIGS. 54-56are exemplary only and not limiting. Various holding fixture and subcardconfigurations having differing number and arrangement of arrays may beused in accordance with the present teachings. Further, the subcardsthat are placed in the holding fixtures need not be of the same sizeand/or arrangement. For example, a subcard having a configuration likethe subcards 5510 may be provided in the same holding fixture as asubcard having a configuration like the subcards 5610. Those havingskill in the art would recognize that other combinations of subcards andconfigurations of subcards and holding fixtures may be used based on thepresent teachings.

In various embodiments, when performing biological testing, differingnumbers of substrates maybe processed in each testing run. For example,one, two, three, four, or more cards may be processed (e.g.,thermocycled) during a given biological testing run. This may beparticularly true in benchtop testing of substrates.

In this situation, it is desirable to achieve substantialchamber-to-chamber thermal uniformity within and between substratesbeing processed. Moreover, it may be desirable to provide information tothe testing instrumentation regarding the number of substrates beingprocessed in a given run.

With reference to FIGS. 57-60, a set of four differing substrate (e.g.,card) carriers are depicted. Each carrier is configured to hold adiffering number of substrates. For example, the carrier 5700 in FIG. 57defines a single cavity 5705 configured to hold a single substrate. Thecarrier 5800 in FIG. 58 defines two cavities 5805 each configured tohold a substrate. The carrier 5900 in FIG. 59 is configured to holdthree substrates in three cavities 5905 defined by the carrier, and thecarrier 6000 in FIG. 60 defines four cavities 6005 configured to holdfour substrates.

Each of the differing carriers in FIGS. 57-60 may include indicatorsreadable by the instrumentation used for processing to indicate how manysubstrates the carrier is configured to hold. By way of example, eachcarrier 5700, 5800, 5900, and 6000 may include one or more fluorescentscan marks 5715, 5815, 5915, and 6015 that indicate how many substratesthe carrier is configured to hold (e.g., 1 scan mark for carrier 5700, 2scan marks for 5800, 3 scan marks for 5900, and 4 scan marks for 6000).The carriers 5700, 5800, 5900, and 6000 also may include an indicator5720, 5820, 5920, and 6020 readable by an individual (e.g., an Arabicnumeral 1 through 4) to indicate how many substrates the carrier isconfigured to hold. Indicators other than Arabic numerals and/orfluorescent scan marks, such as, for example, bar codes and RFIDidentifiers, also may be appropriate identification mechanisms. Thosehaving ordinary skill in the art would recognize numerous ways toachieve identification of the carriers, both by operators and byinstrumentation.

To use the carriers of FIGS. 57-60, a user installs the appropriatenumber of sample-filled substrates (e.g., one to four in the exemplaryembodiments of FIGS. 57-60) into a carrier, places the carrier into thetesting instrumentation, and begins testing (e.g., thermocycling). Thetesting instrumentation reads the indicators on the carrier to determinehow many substrates are being processed and may be configured to controlthe processing steps (such as the thermocycling temperatures, times,locations of applied heat, etc.) based on that information. This mayensure that the testing processes are compatible with the number ofsubstrates being tested so as to promote thermal uniformity.

FIG. 61 depicts another exemplary embodiment of a substrate carrier thatmay be useful when performing biological testing on differing numbers ofsubstrates in a given testing run. The carrier 6100 of FIG. 61 definesfour cavities 6105 that are configured to receive a substrate forbiological testing or a mockup card 6120. The mockup card 6120 may beremovable from a cavity 6105 and replaced with a substrate forbiological testing or may be placed in a cavity 6105 when the cavity6105 is not being used to hold a substrate for biological testing. Eachmockup card 6120 may be provided with an indicator 6115 readable by theinstrumentation used for processing to indicate that a mockup card 6120rather than a sample substrate is in place in a cavity. By being able tosense the number and locations of mockup cards versus sample substratesheld by the carrier 6100 during a testing run, the testing processes maybe adjusted (e.g., thermocycling temperatures, times, and locations ofapplied heat, etc.).

The present teachings provide a variety of structural arrangements,techniques, and/or methodology useful for performing biologicalanalysis, including multiple analyte detection. It should be understoodthat although in some cases the embodiments described herein may focuson a particular aspect, various embodiments may be combined to form asystem and/or substrate configuration useful for multiple analytedetection. By way of example only, various sealing approaches may becombined with various venting approaches. The various embodimentsdescribed herein are not intended to be mutually exclusive.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present invention. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofreported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “less than 10” includes any and allsubranges between (and including) the minimum value of zero and themaximum value of 10, that is, any and all subranges having a minimumvalue of equal to or greater than zero and a maximum value of equal toor less than 10, e.g., 1 to 5.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a layer” includes two or more different layers. As usedherein, the term “include” and its grammatical variants are intended tobe non-limiting, such that recitation of items in a list is not to theexclusion of other like items that can be substituted or added to thelisted items.

Various embodiments of the teachings are described herein. The teachingsare not limited to the specific embodiments described, but encompassequivalent features and methods as known to one of ordinary skill in theart. Other embodiments will be apparent to those skilled in the art fromconsideration of the present specification and practice of the teachingsdisclosed herein. It is intended that the present specification andexamples be considered as exemplary only.

What is claimed is:
 1. A system for distribution of a biological sample,the system comprising: a substrate, wherein the substrate comprises amain fluid supply channel connected to and fluidly communicating with aplurality of sample introduction channels, each sample introductionchannel being connected to and fluidly communicating with a samplechamber, each sample chamber being connected to and fluidlycommunicating with a venting channel; a preloaded reagent contained ineach sample chamber and configured for nucleic acid analysis of abiological sample that enters the substrate; and a sealing instrument,operable to individually isolate each sample chamber from each other soas to substantially prevent sample contained in each sample chamber fromflowing out of each sample chamber, wherein the sealing instrumentcomprises a plurality of protrusions selected from the group consistingof thermal transfer dies, substantially arc-shaped pins, circumferentialdisks and circular pins, each of the plurality of protrusions aligned tointersect and deform the sample introduction or venting channel of eachsample chamber, wherein the substrate is constructed ofdetection-compatible and assay-compatible materials.
 2. The system ofclaim 1, wherein the sealing instrument is configured to be placed incontact with an exterior portion of the substrate.
 3. The system ofclaim 1, wherein the sealing instrument is configured to be placed in atleast one of pressure contact and thermal contact with the substrate. 4.The system of claim 1, wherein the sealing instrument is configured toseal the sample introduction channels and venting channels for eachsample chamber.
 5. The system of claim 1, wherein the sealing instrumentis configured to be placed in contact with the substrate at least one ofbefore a reaction process that occurs in the sample chambers and duringa reaction process that occurs in the sample chambers.
 6. The system ofclaim 5, wherein the sealing instrument is configured to be placed incontact with the substrate before a reaction process that occurs in thesample chambers to seal each sample chamber.
 7. The system of claim 1,wherein the substrate further comprises a film layer and a base, and atleast one venting mechanism configured to permit gas to escape thesubstrate.